RNA Modulating Oligonucleotides with Improved Characteristics for the Treatment of Duchenne and Becker Muscular Dystrophy

ABSTRACT

The current invention provides an improved oligonucleotide and its use for treating, ameliorating, preventing and/or delaying DMD or BMD.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/201,951, filed Nov. 27, 2018, which is a continuation of U.S.application Ser. No. 15/232,493, filed Aug. 9, 2016, now U.S. Pat. No.10,179,912, which is a continuation of U.S. application Ser. No.14/444,244, filed Jul. 28, 2014, which is a continuation ofPCT/NL2013/050045, filed Jan. 28, 2013, which claims priority toEP12152934.1, filed Jan. 27, 2012 and U.S. provisional application Nos.61/612,467, filed Mar. 19, 2012, and 61/591,354, filed Jan. 27, 2012.The disclosures of the above applications are each incorporated byreference in their entirety.

SEQUENCE LISTING

This application is being filed with a Sequence Listing in ComputerReadable Format (CRF) entitled “0105_10_US1CN3_SL.txt” having 214794bytes, created on Dec. 15, 2020, which is incorporated herein byreference in its entirety.

FIELD

The invention relates to the field of human genetics, more specificallyneuromuscular disorders. The invention in particular relates to the useof an oligonucleotide with improved characteristics enhancing clinicalapplicability as further defined herein.

BACKGROUND OF THE INVENTION

Neuromuscular diseases are characterized by impaired functioning of themuscles due to either muscle or nerve pathology (myopathies andneuropathies). The myopathies include genetic muscular dystrophies thatare characterized by progressive weakness and degeneration of skeletal,heart and/or smooth muscle. Duchenne muscular dystrophy (DMD) and Beckermuscular dystrophy (BMD) are the most common childhood forms of musculardystrophy. DMD is a severe, lethal neuromuscular disorder resulting in adependency on wheelchair support before the age of 12 and patients oftendie before the age of thirty due to respiratory- or heart failure. It iscaused by reading frame-shifting deletions (˜67%) or duplications (˜7%)of one or more exons, or by point mutations (˜25%) in the 2.24 Mb DMDgene, resulting in the absence of functional dystrophin. BMD is alsocaused by mutations in the DMD gene, but these maintain the open readingframe, yield semi-functional dystrophin proteins, and result in atypically much milder phenotype and longer lifespan. During the lastdecade, specific modification of splicing in order to restore thedisrupted reading frame of the transcript has emerged as a promisingtherapy for DMD (van Ommen et al., 2008; Yokota et al., 2007; vanDeutekom et al., 2007; Goemans et al., 2011; Cirak et al., 2011). Usinghighly sequence-specific antisense oligonucleotides (AONs) which bind tothe exon flanking or containing the mutation and which interfere withits splicing signals, the skipping of that exon can be induced duringthe processing of the DMD pre-mRNA. Despite the resulting truncatedtranscript, the open reading frame is restored and a protein isintroduced which is similar to those found in BMD patients. AON-inducedexon skipping provides a mutation-specific, and thus personalized,therapeutic approach for DMD patients. Several oligonucleotides arecurrently being developed for skipping most relevant exons of thedystrophin pre-mRNA such as exons 2, 8, 9, 17, 29, 43, 44, 45, 46, 47,48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60-63, 71-78 as described inWO 02/024906, WO2004/083446, WO2006/112705, WO2007/135105, WO2009/139630, WO 2010/050801 or WO 2010/050802.

As the majority of the mutations cluster around exons 45 to 55, theskipping of one specific exon may be therapeutic for many patients withdifferent mutations. The skipping of exon 51 applies to the largestsubset of patients (˜13%), including those with deletions of exons 45 to50, 48 to 50, 50, or 52. The AONs applied are chemically modified toresist endonucleases, exonucleases and RNaseH, and to promote RNAbinding and duplex stability. Two different AON chemistries arecurrently being developed for exon 51 skipping in DMD: 2′-O-methylphosphorothioate RNA AONs (2OMePS, GSK2402968/PRO051) andphosphorodiamidate morpholino oligomers (PMO, AVI-4658) (Goemans et al.,2011; Cirak et al., 2011). In two independent phase I/II studies, bothwere shown to specifically induce exon 51 skipping and at least partlyrestore dystrophin expression at the muscle fiber membranes aftersystemic administration. Although AONs are typically not well taken upby healthy muscle fibers, the dystrophin deficiency in DMD, resulting indamaged and thus more permeable fiber membranes, actually promotesuptake. In studies in the dystrophin-deficient mdx mouse model,2′-O-methyl phosphorothioate RNA oligonucleotides have demonstrated anup to 10 times higher uptake in different muscle groups when compared tothat in wild type mice (Heemskerk et al., 2010). Although the recentphase I/II results with both 2′-O-methyl phosphorothioate RNA andphosphorodiamidate morpholino AONs in DMD patients confirm this enhanceduptake in dystrophic muscle, the different chemical modifications seemedto result in a differential uptake by and distribution through muscle.The levels of novel dystrophin in both studies after 3 months oftreatment were promising but still moderate and challenges the field toinvestigate next generation oligochemistry.

The particular characteristics of a chosen chemistry at least in partaffects the delivery of an AON to the target transcript: administrationroute, biostability, biodistribution, intra-tissue distribution, andcellular uptake and trafficking. In addition, further optimization ofoligonucleotide chemistry is conceived to enhance binding affinity andstability, enhance activity, improve safety, and/or to reduce cost ofgoods by reducing length or improving synthesis and/or purificationprocedures. Multiple chemical modifications have become generally and/orcommercially available to the research community (such as 2′-O-methylRNA and 5-substituted pyrimidines and 2,6-diaminopurines), whereas mostothers still present significant synthetic effort to obtain. Especiallypreliminary encouraging results have been obtained using 2′-O-methylphosphorothioate RNA containing modifications on the pyrimidine andpurine bases as identified herein.

In conclusion, to enhance the therapeutic applicability of AONs for DMD,there is a need for AONs with further improved characteristics.

DESCRIPTION OF THE INVENTION Oligonucleotide

In a first aspect, the invention provides an oligonucleotide comprisinga 2′-O-methyl RNA monomer and a phosphorothioate backbone or consistingof 2′-O-methyl RNA monomers linked by phosphorothioate backbones, andcomprising a 5-methylpyrimidine and/or a 2,6-diaminopurine basepreferably for use as a medicament for treating Duchenne MuscularDystrophy or Becker Muscular Dystrophy.

Therefore, the invention provides an oligonucleotide comprising a2′-O-methyl RNA monomer, a phosphorothioate backbone and a5-methylpyrimidine and/or a 2,6-diaminopurine base preferably for use asa medicament for treating Duchenne Muscular Dystrophy or Becker MuscularDystrophy.

Accordingly, the invention also provides an oligonucleotide consistingof 2′-O-methyl RNA monomers and a phosphorothioate backbone andcomprises a 5-methylpyrimidine and/or a 2,6-diaminopurine basepreferably for use as a medicament for treating Duchenne MuscularDystrophy or Becker Muscular Dystrophy.

It is clear for the skilled person that “an RNA monomer” as present inan oligonucleotide of the invention may also be identified as being “anRNA nucleotide residue”. Both terms may be used interchangeablythroughout the application.

Within the context of the invention, “a” in each of the followingexpressions means “at least one”: a 2′-O-methyl RNA monomer, a2′-O-methyl RNA nucleotide residue, a 2′-O-methyl phosphorothioate RNAmonomer, a 5-methylpyrimidine base, a 2,6-diaminopurine base.

Within the context of the invention, it is clear for the skilled personthat “an oligonucleotide comprising a 2′-O-methyl RNA monomer, aphosphorothioate backbone” could be replaced by “an oligonucleotidecomprising a 2′-O-methyl RNA monomer linked by phosphorothioatebackbones”. The same holds for “an oligonucleotide consisting of2′-O-methyl RNA monomers and a phosphorothioate backbone” that could bereplaced by “an oligonucleotide consisting of 2′-O-methyl RNA monomerlinked by phosphorothioate backbones”.

In the context of the invention, the expression “for use as a medicamentfor treating Duchenne Muscular Dystrophy or Becker Muscular Dystrophy”could be replaced by the expression “for use in the treatment ofDuchenne Muscular Dystrophy or Becker Muscular Dystrophy.”

Preferably, an oligonucleotide is an oligonucleotide with less than 34nucleotides. Said oligonucleotide may have 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides. Such oligonucleotide may also be identified as anoligonucleotide having from 10 to 33 nucleotides.

Accordingly, an oligonucleotide of the invention comprises a 2′-O-methylRNA monomer and a phosphorothioate backbone and comprises less than 34nucleotides (i.e., it comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides).

Accordingly, an oligonucleotide of the invention consists of 2′-O-methylRNA monomers linked by phosphorothioate backbone and comprises less than34 nucleotides (i.e., it comprises 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides).

Accordingly, an oligonucleotide of the invention comprises a 2′-O-methylRNA monomer, a phosphorothioate backbone, comprises less than 34nucleotides (i.e., it comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides)and a 5-methylpyrimidine and/or a 2,6-diaminopurine base.

Accordingly, an oligonucleotide of the invention consists of 2′-O-methylRNA monomers linked by phosphorothioate backbone, and comprises lessthan 34 nucleotides (i.e., it comprises 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides) and a 5-methylpyrimidine and/or a 2,6-diaminopurine base.

Each of these oligonucleotides is for use or may be for use as amedicament for treating Duchenne Muscular Dystrophy or Becker MuscularDystrophy.

An oligonucleotide of the invention comprises or consists of a2′-O-methyl phosphorothioate RNA monomer. Such oligonucleotide comprisesa 2′-O-methyl RNA monomer connected through or linked by aphosphorothioate backbone or consists of 2′-O-methyl phosphorothioateRNA. Preferably, such oligonucleotide consists of a 2′-O-methylphosphorothioate RNA. Such chemistry is known to the skilled person.Throughout the application, an oligonucleotide comprising a 2′-O-methylRNA monomer and a phosphorothioate backbone may be replaced by anoligonucleotide comprising a 2′-O-methyl phosphorothioate RNA.Throughout the application, an oligonucleotide consisting of 2′-O-methylRNA monomers linked by or connected through phosphorothioate backbonesmay be replaced by an oligonucleotide consisting of 2′-O-methylphosphorothioate RNA.

In the context of the invention, “backbone” is used to identify thelinkage between two sugar units or modified versions of a sugar unit ormoiety as later defined herein (i.e. internucleoside linkage).Throughout the description, the words “backbone”, “internucleosidelinkage” and “linkage” may be used interchangeably. Thus, anoligonucleotide having 10 nucleotides contains 9 backbones, linking the10 sugar units or modified versions of a sugar unit or moiety as laterdefined herein together. At least one of the backbones of theoligonucleotide according to the invention consists of aphosphorothioate moiety, linking two sugar units or modified versions ofa sugar unit or moiety as later defined herein. Thus, at least onephosphodiester backbones present in RNA is replaced by phosphorothioatemoiety. A naturally occurring internucleoside linkage or backbone is the3′ to 5′ phosphodiester linkage.

In addition, an oligonucleotide of the invention may comprise a basemodification that increases binding affinity to target strands,increases melting temperature of the resulting duplex of saidoligonucleotide with its target, and/or decreases immunostimulatoryeffects, and/or increases biostability, and/or improves biodistributionand/or intra-tissue distribution, and/or cellular uptake andtrafficking. In a more preferred embodiment, an oligonucleotide of theinvention comprises a 5-methylpyrimidine and/or a 2,6-diaminopurinebase. A 5-methylpyrimidine base is selected from a 5-methylcytosineand/or a 5-methyluracil and/or a thymine, in which thymine is identicalto 5-methyluracil.

Accordingly, the expression “comprises a 5-methylcytosine and/or a5-methyluracil and/or a 2,6-diaminopurine base” in the context of themodified oligonucleotide of the invention may be replaced by “comprisesa base modification selected from the group consisting of: a5-methylcytosine, a 5-methyluracil and a 2,6-diaminopurine base”.

Where an oligonucleotide of the invention has two or more such basemodifications, said base modifications may be identical, for example allsuch modified bases in the oligonucleotide are 5-methylcytosine, or saidbase modifications may be combinations of different base modifications,for example the oligonucleotide may have one or more 5-methylcytosinesand one or more 5-methyluracils.

‘Thymine’ and ‘5-methyluracil’ may be interchanged throughout thedocument. In analogy, 2,6-diaminopurine is identical to 2-aminoadenineand these terms may be interchanged throughout the document. The use of2,6-diaminopurine has been disclosed in another context in U.S. Pat. No.7,745,420.

The term “base modification” or “modified base” as identified hereinrefers to the modification of an existing base (i.e., pyrimidine orpurine base) or to the de novo synthesis of a base. This de novosynthetized base could be qualified as “modified” by comparison to anexisting base. An oligonucleotide of the invention comprising a5-methylcytosine and/or a 5-methyluracil and/or a 2,6-diaminopurine basemeans that at least one of the cytosine nucleobases of saidoligonucleotide has been modified by substitution of the proton at the5-position of the pyrimidine ring with a methyl group, i.e., a5-substituted cytosine, and/or that at least one of the uracilnucleobases of said oligonucleotide has been modified by substitution ofthe proton at the 5-position of the pyrimidine ring with a methyl group(i.e., a 5-methyluracil), and/or that at least one of the adeninenucleobases of said oligonucleotide has been modified by substitution ofthe proton at the 2-position with an amino group (i.e., a2,6-diaminopurine), respectively. Within the context of the invention,the expression “the substitution of a proton with a methyl group inposition 5 of the pyrimidine ring” may be replaced by the expression“the substitution of a pyrimidine with a 5-methylpyrimidine,” withpyrimidine referring to only uracil, only cytosine or both. Likewise,within the context of the invention, the expression “the substitution ofa proton with an amino group in position 2 of adenine” may be replacedby the expression “the substitution of an adenine with a2,6-diaminopurine.” If said oligonucleotide comprises 1, 2, 3, 4, 5, 6,7, 8, 9 or more cytosines, uracils, and/or adenines, at least one, 2, 3,4, 5, 6, 7, 8, 9 or more cytosines, uracils and/or adenines respectivelyhave been modified this way. Preferably all cytosines, uracils and/oradenines have been modified this way or substituted by 5-methylcytosine,5-methyluracil and/or 2,6-diaminopurine, respectively. No need to saythat this aspect of the invention could only be applied tooligonucleotides comprising at least one cytosine, uracil, or adenine,respectively, in their sequence. An oligonucleotide comprising at leastone 5-methylcytosine, 5-methyluracil and/or 2,6-diaminopurine may becalled a modified oligonucleotide by reference to its non-modifiedcounterpart comprising no 5-methylcytosine, no 5-methyluracil and no2,6-diaminopurine. A non-modified counterpart may also be identified asbeing an oliognucleotide comprising unmodified cytosines, unmodifieduraciles and unmodified adenines. Preferred non-modified sequences arerepresented by one of the following base or nucleotide sequencescomprising or consisting of SEQ ID NO:91, 93-170.

We discovered that the presence of a 5-methylcytosine, 5-methyluraciland/or a 2,6-diaminopurine in an oligonucleotide of the invention has apositive effect on at least one of the parameters of saidoligonucleotides. In this context, parameters may include: bindingaffinity and/or kinetics, exon skipping activity, biostability,(intra-tissue) distribution, cellular uptake and/or trafficking, and/orimmunogenicity of said oligonucleotide, as explained below. Saidpositive effect may be correlated with the number or percentage of basemodifications incorporated. For the parameter of exon skipping activity,we found for some oligonucleotides that modification of nucleobases isnot needed per se to obtain relatively high levels of exon skipping.This may be related to the specific role (and strength) of thespecifically targeted sequence within the exon in its splicing process.

Binding affinity and kinetics depend on the AON's thermodynamicproperties. These are at least in part determined by the meltingtemperature of said oligonucleotide (Tm; calculated with, e.g., theoligonucleotide properties calculator(www.unc.edu/˜cail/biotool/oligo/index.html oreu.idtdna.com/analyzer/Applications/OligoAnalyzer/) for single strandedRNA using the basic Tm and the nearest neighbor model), and/or the freeenergy of the oligonucleotide-target exon complex (using RNA structureversion 4.5 or RNA mfold version 3.5). If a Tm is increased, the exonskipping activity typically increases, but when a Tm is too high, theAON is expected to become less sequence-specific. An acceptable Tm andfree energy depend on the sequence of the oligonucleotide. Therefore, itis difficult to give preferred ranges for each of these parameters.

Exon skipping activity is preferably measured by analysing total RNAisolated from AON-treated muscle cell cultures or muscle tissue byreverse transcriptase polymerase chain reaction (RT-PCR) using DMDgene-specific primers flanking the targeted exon as described(Aartsma-Rus et al., 2003). RT-PCR products are analyzed on 1-2% agarosegels or with the Agilent 2100 bioanalyzer (Agilent Technologies, TheNetherlands). The ratio of shorter transcript fragments, representingtranscripts in which the targeted exon is skipped, to the total oftranscript products is assessed (calculated as percentage of exonskipping induced by an AON). Shorter fragments may also be sequenced todetermine the correctness and specificity of the targeted exon skipping.An increase in percentage of exon skipping may be detected for amodified oligonucleotide of the invention (i.e., an oligonucleotidecomprising a 2′-O-methyl RNA monomer, a phosphorothioate backbone and a5-methylpyrimidine and/or a 2,6-diaminopurine base) compared to itsnon-modified counterpart (i.e., an oligonucleotide comprising a2′-O-methyl RNA monomer, a phosphorothioate backbone and not comprisingany 5-methylpyrimidine and any 2,6-diaminopurine base). Said increase ispreferably a detectable increase assessed as explained above usingRT-PCR. Said increase is preferably an increase of at least 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%,150%, 160%, 170%, 180%, 190%, 200%, 210%, 300%, 400%, 500%, 600%, 700%,800%, 900%, 1000%, or at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 timeshigher, or even 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times higher ormore.

Biodistribution and biostability are preferably at least in partdetermined by a validated hybridization ligation assay adapted from Yuet al., 2002. In an embodiment, plasma or homogenized tissue samples areincubated with a specific capture oligonucleotide probe. Afterseparation, a DIG-labeled oligonucleotide is ligated to the complex anddetection followed using an anti-DIG antibody-linked peroxidase.Non-compartmental pharmacokinetic analysis is performed using WINNONLINsoftware package (model 200, version 5.2, Pharsight, Mountainview,Calif.). Levels of AON (ug) per mL plasma or mg tissue are monitoredover time to assess area under the curve (AUC), peak concentration(C_(max)), time to peak concentration (T_(max)), terminal half life andabsorption lag time (t_(lag)). Such a preferred assay has been disclosedin the experimental part.

AONs may stimulate an innate immune response by activating the Toll-likereceptors (TLR), including TLR9 and TLR7 (Krieg et al., 1995). Theactivation of TLR9 typically occurs due to the presence ofnon-methylated CG sequences present in oligodeoxynucleotides (ODNs), bymimicking bacterial DNA which activates the innate immune system throughTLR9-mediated cytokine release. The 2′-O-methyl modification is howeversuggested to markedly reduce such possible effect. TLR7 has beendescribed to recognize uracil repeats in RNA (Diebold et al., 2006).Activation of TLR9 and TLR7 result in a set of coordinated immuneresponses that include innate immunity (macrophages, dendritic cells(DC), and NK cells) (Krieg et al., 1995; Krieg, 2000). Several chemo-and cytokines, such as IP-10, TNFα, IL-6, MCP-1 and IFNα (Wagner, 1999;Popovic et al., 2006) have been implicated in this process. Theinflammatory cytokines attract additional defensive cells from theblood, such as T and B cells. The levels of these cytokines can beinvestigated by in vitro testing. In short, human whole blood isincubated with increasing concentrations of AONs after which the levelsof the cytokines are determined by standard commercially available ELISAkits. Such a preferred assay has been described in the experimentalpart. A decrease in immunogenicity preferably corresponds to adetectable decrease of concentration of at least one of the cytokinesmentioned above by comparison to the concentration of correspondingcytokine in an assay in a cell treated with an oligonucleotidecomprising at least one 5-methylcytosine compared to a cell treated witha corresponding oligonucleotide having no 5-methylcytosines.

Accordingly, a preferred oligonucleotide of the invention has animproved parameter, such as an acceptable or a decreased immunogenicityand/or a better biodistribution and/or acceptable or improved RNAbinding kinetics and/or thermodynamic properties by comparison to acorresponding oligonucleotide consisting of a 2′-O-methylphosphorothioate RNA without a 5-methylcytosine, a 5-methyluracil and a2,6-diaminopurine (i.e. so called non-modified oligonucleotide). Saidnon-modified oligonucleotide may also be identified as being anoliognucleotide comprising unmodified cytosines, unmodified uraciles andunmodified adenines. Each of these parameters could be assessed usingassays known to the skilled person or preferably as disclosed herein.

Below other chemistries and modifications of the oligonucleotide of theinvention are defined. These additional chemistries and modificationsmay be present in combination with the chemistry already defined forsaid oligonucleotide, i.e. the presence of a 5-methylcytosine, a5-methyluracil and/or a 2,6-diaminopurine, and the oligonucleotidecomprising or consisting of a 2′-methyl phosphorothioate RNA.

A preferred oligonucleotide of the invention comprises or consists of anRNA molecule or a modified RNA molecule. In a preferred embodiment, anoligonucleotide is single stranded. The skilled person will understandthat it is however possible that a single stranded oligonucleotide mayform an internal double stranded structure. However, thisoligonucleotide is still named a single stranded oligonucleotide in thecontext of this invention.

In addition to the modifications described above, the oligonucleotide ofthe invention may comprise further modifications such as different typesof nucleic acid monomers or nucleotides as described below. Differenttypes of nucleic acid monomers may be used to generate anoligonucleotide of the invention. Said oligonucleotide may have at leastone backbone, and/or sugar modification and/or at least one basemodification compared to an RNA-based oligonucleotide.

A base modification includes a modified version of the natural purineand pyrimidine bases (e.g. adenine, uracil, guanine, cytosine, andthymine), such as hypoxanthine, orotic acid, agmatidine, lysidine,2-thiopyrimidine (e.g. 2-thiouracil, 2-thiothymine), G-clamp and itsderivatives, 5-substituted pyrimidine (e.g. 5-halouracil,5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil,5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine,Super T), 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine,8-aza-7-deazaguanine, 8-aza-7-deazaadenine,8-aza-7-deaza-2,6-diaminopurine, Super G, Super A, and N4-ethylcytosine,or derivatives thereof; N²-cyclopentylguanine (cPent-G),N²-cyclopentyl-2-aminopurine (cPent-AP), and N²-propyl-2-aminopurine(Pr-AP), pseudouracil or derivatives thereof; and degenerate oruniversal bases, like 2,6-difluorotoluene or absent bases like abasicsites (e.g. 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose;or pyrrolidine derivatives in which the ring oxygen has been replacedwith nitrogen (azaribose)). Examples of derivatives of Super A, Super Gand Super T can be found in U.S. Pat. No. 6,683,173 (Epoch Biosciences),which is incorporated here entirely by reference. cPent-G, cPent-AP andPr-AP were shown to reduce immunostimulatory effects when incorporatedin siRNA (Peacock H. et al. J. Am. Chem. Soc. 2011, 133, 9200).

A pseudouracil is a naturally occurring isomerized version of uracil,with a C-glycoside rather than the regular N-glycoside as in uridine.Pseudouridine-containing synthetic mRNA may have an improved safetyprofile compared to uridine-containing mRNA (WO 2009127230, incorporatedhere in its entirety by reference).

In an embodiment, an oligonucleotide of the invention comprises anabasic site or an abasic monomer. Within the context of the invention,such monomer may be called an abasic site or an abasic monomer. Anabasic monomer or abasic site is a monomer or building block that lacksa nucleobase by comparison to a corresponding monomer comprising anucleobase. Within the invention, an abasic monomer is thus a buildingblock part of an oligonucleotide but lacking a nucleobase. Such abasicmonomer may be present or linked or attached or conjugated to a freeterminus of an oligonucleotide. In a more preferred embodiment, anoligonucleotide of the invention comprises 1-20 or more abasic monomers.Therefore, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or more abasic monomers may be present in an oligonucleotideof the invention.

An abasic monomer may be of any type known and conceivable by theskilled person, non-limiting examples of which are depicted below:

Herein, R₁ and R₂ are independently H, an oligonucleotide or otherabasic site(s), provided that not both R₁ and R₂ are H and R₁ and R₂ arenot both an oligonucleotide. An abasic monomer(s) can be attached toeither or both termini of the oligonucleotide as specified before. Itshould be noted that an oligonucleotide attached to one or two an abasicsite(s) or abasic monomer(s) may comprise less than 10 nucleotides. Inthis respect, the oligonucleotide according to the invention maycomprise at least 10 nucleotides, optionally including one or moreabasic sites or abasic monomers at one or both termini.

Depending on its length an oligonucleotide of the invention may comprise1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 basemodifications. It is also encompassed by the invention to introduce morethan one distinct base modification in said oligonucleotide.

A sugar modification includes a modified version of the ribosyl moiety,such as 2′-0-modified RNA such as 2′-O-alkyl or 2′-O-(substituted)alkyle.g. 2′-O-methyl, 2′-O-(2-cyanoethyl), 2′-O-(2-methoxy)ethyl (2′-MOE),2′-O-(2-thiomethyl)ethyl, 2′-O-butyryl, 2′-O-propargyl, 2′-O-allyl,2′-O-(3-amino)propyl, 2′-O-(3-(dimethylamino)propyl),2′-O-(2-amino)ethyl, 2′-O-(2-(dimethylamino)ethyl); 2′-deoxy (DNA);2′-O-(haloalkoxy)methyl (Arai K. et al. Boorg. Med. Chem. 2011, 21,6285) e.g. 2′-O-(2-chloroethoxy)methyl (MCEM),2′-O-(2,2-dichloroethoxy)methyl (DCEM); 2′-O-alkoxycarbonyl e.g.2′-O-[2-(methoxycarbonyl)ethyl] (MOCE),2′-O-[2-(N-methylcarbamoyl)ethyl] (MCE),2′-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DCME); 2′-halo e.g. 2′-F, FANA(2′-F arabinosyl nucleic acid); carbasugar, sulfa and sulfosugar andazasugar modifications; 3′-O-alkyl e.g. 3′-O-methyl, 3′-O-butyryl,3′-O-propargyl; 4′-carboxy e.g. 4′-carboxythymidine (Hari et al.); andtheir derivatives.

Other sugar modification includes “bridged” or “bicylic” nucleic acid(BNA), e.g. locked nucleic acid (LNA), xylo-LNA, α-L-LNA, β-D-LNA, cEt(2′-O,4′-C constrained ethyl) LNA, cMOEt (2′-O,4′-C constrainedmethoxyethyl) LNA, ethylene-bridged nucleic acid (ENA), tricyclo DNA(tcDNA, tc-PS-DNA e.g. US patent application 20120149756);3′-S-phosphorothiolate DNA (e.g. Org. Biol. Chem. 2013, 11, 966); doublyconstrained nucleic acid (tri-NA, e.g. Hanessian et al.); unlockednucleic acid (UNA); cyclohexenyl nucleic acid (CeNA), altriol nucleicacid (ANA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA),pyranosyl-RNA (p-RNA), 3′-deoxypyranosyl-DNA (p-DNA); morpholino (ase.g. in PMO, PPMO, PMOPlus, PMO-X); and their derivatives. Depending onits length, an oligonucleotide of the invention may comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30 sugar modifications. It is also encompassedby the invention to introduce more than one distinct sugar modificationin said oligonucleotide. In an embodiment, an oligonucleotide as definedherein comprises or consists of an LNA or a derivative thereof. BNAderivatives are for example described in WO 2011/097641, which isincorporated in its entirety by reference. In a more preferredembodiment, an oligonucleotide of the invention is fully 2′-O-methylmodified. Examples of PMO-X are described in WO2011150408, which isincorporated here in its entirety by reference.

A backbone modification includes a modified version of thephosphodiester present in RNA, such as phosphorothioate (PS), chirallypure phosphorothioate, phosphorodithioate (PS2), phosphonoacetate(PACE), phosphonoacetamide (PACA), thiophosphonoacetate,thiophosphonoacetamide, phosphorothioate prodrug, H-phosphonate, methylphosphonate, methyl phosphonothioate, methyl phosphate, methylphosphorothioate, ethyl phosphate, ethyl phosphorothioate,boranophosphate, boranophosphorothioate, methyl boranophosphate, methylboranophosphorothioate, methyl boranophosphonate, methylboranophosphonothioate, and their derivatives. Another modificationincludes phosphoramidite, phosphoramidate, N3′→P5′ phosphoramidate,phosphordiamidate, phosphorothiodiamidate, sulfamate,dimethylenesulfoxide, sulfonate, triazole, oxalyl, carbamate,methyleneimino (MMI), 3′-S-phosphorothiolate (Org. Biol. Chem. 2013, 11,966) and thioacetamido nucleic acid (TANA); and their derivatives.Depending on its length, an oligonucleotide of the invention maycomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 backbonemodifications. It is also encompassed by the invention to introduce morethan one distinct backbone modification in said oligonucleotide.

In a preferred embodiment, an oligonucleotide of the invention comprisesat least one phosphorothioate modification. In a more preferredembodiment, an oligonucleotide of the invention is fullyphosphorothioate modified.

Other chemical modifications of an oligonucleotide of the inventioninclude peptide-base nucleic acid (PNA), boron-cluster modified PNA,pyrrolidine-based oxy-peptide nucleic acid (POPNA), glycol- orglycerol-based nucleic acid (GNA), threose-based nucleic acid (TNA),acyclic threoninol-based nucleic acid (aTNA), morpholino-basedoligonucleotide (PMO, PPMO, PMO-X), cationic morpholino-based oligomers(PMOPlus), oligonucleotides with integrated bases and backbones (ONIBs),pyrrolidine-amide oligonucleotides (POMs); and their derivatives.

In another embodiment, an oligonucleotide comprises a peptide nucleicacid and/or a morpholino phosphorodiamidate or a derivative thereof.

In another embodiment, an oligonucleotide comprises a monothiophosphatemoiety at the 5′ position of the 5′ terminal residue and/or amonothiophosphate moiety at the 3′ position of the 3′ terminal residue.These monothiophosphate groups have been shown to improveoligonucleotide stability (e.g., US patent application20120148664—miRagen).

With the advent of nucleic acid mimicking technology it has becomepossible to generate molecules that have a similar, preferably the samehybridization characteristics in kind not necessarily in amount asnucleic acid itself. Such functional equivalents are of course alsosuitable for use in the invention.

The skilled person will understand that not each sugar, base, and/orbackbone may be modified the same way. Several distinct modified sugars,bases and/or backbones may be combined into one single oligonucleotideof the invention.

A person skilled in the art will also recognize that there are manysynthetic derivatives of oligonucleotides. A backbone modificationincludes a modified version of the phosphodiester present in RNA, suchas phosphorothioate (PS), chirally pure phosphorothioate,phosphorodithioate (PS2), phosphonoacetate (PACE), phosphonoacetamide(PACA), thiophosphonoacetate, thiophosphonoacetamide, phosphorothioateprodrug, H-phosphonate, methyl phosphonate, methyl phosphonothioate,methyl phosphate, methyl phosphorothioate, ethyl phosphate, ethylphosphorothioate, boranophosphate, boranophosphorothioate, methylboranophosphate, methyl boranophosphorothioate, methylboranophosphonate, methyl boranophosphonothioate, and their derivatives.Another modification includes phosphoramidite, phosphoramidate, N3′→P5′phosphoramidate, phosphordiamidate, phosphorothiodiamidate, sulfamate,dimethylenesulfoxide, sulfonate, and thioacetamido nucleic acid (TANA);and their derivatives.

Preferably, said oligonucleotide comprises RNA, as RNA/RNA duplexes arevery stable. It is preferred that an RNA oligonucleotide comprises amodification providing the RNA with an additional property, for instanceresistance to endonucleases, exonucleases, and RNaseH, additionalhybridisation strength, increased stability (for instance in a bodilyfluid), increased or decreased flexibility, increased activity, reducedtoxicity, increased intracellular transport, tissue-specificity, etc. Inaddition, the mRNA complexed with the oligonucleotide of the inventionis preferably not susceptible to RNaseH cleavage. Preferredmodifications have been identified above.

Accordingly, the invention provides an oligonucleotide comprising a2′-O-methyl phosphorothioate RNA monomer or consisting of 2′-O-methylphosphorothioate RNA and comprising a 5-methylpyrimidine and/or a2,6-diaminopurine base. Most preferably, this oligonucleotide consistsof 2′-O-methyl RNA monomers connected through a phosphorothioatebackbone and all of its cytosines and/or all of its uracils and/or allof its adenines, independently, have been substituted by5-methylcytosine, 5-methyluracil and/or 2,6-diaminopurine, respectively.Preferred modified and non-modified oligonucleotides encompassed by theinvention and disclosed herein comprises or consists of one of a base ornucleotide sequence selected from one of SEQ ID NO: 14-90 as identifiedin table 1. The expression “oligonucleotide represented by a nucleotideor base sequence selected from SEQ ID NO:14-90” could be replaced by theexpression “oligonucleotide represented by a nucleotide or base sequenceselected from one of SEQ ID NO:14-90” or by the expression“oligonucleotide represented by a nucleotide or base sequence selectedfrom the list of SEQ ID NO:14-90”. The same holds for other groups ofSEQ ID NO referred herein.

Preferred non-modified oligonucleotides are derived from one of SEQ IDNO:14-90 and encompassed by the present invention and disclosed hereincomprises or consists of one of a base or nucleotide sequences selectedfrom SEQ ID NO: 91, 93-170.

Modified oligonucleotides are preferably derived from one of SEQ IDNO:14-90 and encompassed by the present invention and disclosed hereincomprises or consists of one of a base or nucleotide sequences selectedfrom SEQ ID NO: 92, 171-213, 215.

Please note that two SEQ ID NO present in the sequence listing areidentical: SEQ ID NO:91 is identical with SEQ ID NO: 132. SEQ ID NO: 92is identical with SEQ ID NO:199.

The sequence representing each of these oligonucleotides is disclosed inTables 1-3 and in the sequence listing. Later on in the description,most preferred oligonucleotides are described in more detail.

Thus, an oligonucleotide of the invention may have:

At least one and preferably all cytosines substituted with5-methylcytosines,

At least one and preferably all cytosines substituted with5-methylcytosines and at least one and preferably all uracilssubstituted with 5-methyluracils,

At least one and preferably all cytosines substituted with5-methylcytosines and at least one and preferably all adeninessubstituted with 2,6-diaminopurines,

At least one and preferably all cytosines substituted with5-methylcytosines and at least one and preferably all uracilssubstituted with 5-methyluracils and at least one and preferably alladenines substituted with 2,6-diaminopurines,

At least one and preferably all uracils substituted with5-methyluracils,

At least one and preferably all uracils substituted with 5-methyluracilsand at least one and preferably all adenines substituted with2,6-diaminopurines, or

At least one and preferably all adenines substituted with2,6-diaminopurines.

However, an oligonucleotide may also have at least one or at least twoor at least half or all its cytosines substituted with5-methylcytosines. If a non-modified oligonucleotide of the inventionpreferably based on SEQ ID NO:14-90 has x cytosines, x being an integerranged from 1 to 33, a corresponding modified oligonucleotide of theinvention may have 1, 2, 3, . . . (x−2), (x−1), x 5-methylcytosines.

If x is 3 in such a non-modified oligonucleotide, the number of5-methylcytosines in a corresponding modified oligonucleotide is 1, 2 or3.

If x is 4 in such a non-modified oligonucleotide, the number of5-methylcytosines in a corresponding modified oligonucleotide is 1, 2, 3or 4.

If x is 5 in such a non-modified oligonucleotide, the number of5-methylcytosines in a corresponding modified oligonucleotide is 1, 2,3, 4 or 5.

If x is 6 in such a non-modified oligonucleotide, the number of5-methylcytosines in a corresponding modified oligonucleotide is 1, 2,3, 4, 5 or 6.

If x is 7 in such a non-modified oligonucleotide, the number of5-methylcytosines in a corresponding modified oligonucleotide is 1, 2,3, 4, 5, 6 or 7.

If x is 8 in such a non-modified oligonucleotide, the number of5-methylcytosines in a corresponding modified oligonucleotide is 1, 2,3, 4, 5, 6, 7, or 8.

The same holds for uracils substituted with 5-methyluracils and adeninessubstituted with 2,6-diaminopurines.

Preferably, an oligonucleotide of the invention is for use as amedicament for DMD, more preferably said oligonucleotide is for use intherapeutic RNA modulation. Therefore, an oligonucleotide is anantisense oligonucleotide (AON). An antisense oligonucleotide is anoligonucleotide which is reverse complementary to a specific sequence ofthe DMD or dystrophin pre-mRNA derived from the coding sense strand of aDNA of an individual. This oligonucleotide binds to and/or targetsand/or hybridizes and/or is able to bind to and/or is able to targetand/or is able to hybridize said sequence of said pre-mRNA. Theobjective of RNA modulation for DMD is to skip one or more specificexons in the DMD or dystrophin pre-mRNA in order to restore the openreading frame of the transcript and to induce the expression of ashorter but (more) functional dystrophin protein, with the ultimate goalto be able to interfere with the course of the disease.

In a preferred embodiment, an oligonucleotide of the invention is thusused for inducing exon-skipping in the DMD or dystrophin pre-mRNA in acell, in an organ, in a tissue and/or in an individual. Exon-skippingresults in a mature DMD or dystrophin mRNA that does not contain askipped exon and thus, when said exon codes for amino acids, can lead tothe expression of a shorter protein product. The skipping of an exon ispreferably induced by the binding of an AON to specific exon-internalsequences comprising splicing regulatory elements, the splice sitesand/or intronic branchpoint sequences.

As defined herein a DMD pre-mRNA preferably means a pre-mRNA of a DMDgene coding for a dystrophin protein. A mutated DMD pre-mRNA correspondsto a pre-mRNA of a BMD or DMD patient with a mutation when compared to awild type DMD pre-mRNA of a non-affected person, resulting in (reducedlevels of) an aberrant protein (BMD), or the absence of functionaldystrophin (DMD). A DMD pre-mRNA is also named a dystrophin pre-mRNA. ADMD gene may also be named a dystrophin gene. Dystrophin and DMD may beused interchangeably throughout the application.

A patient is preferably intended to mean a patient having DMD or BMD aslater defined herein or a patient susceptible to develop DMD or BMD dueto his or her genetic background. In the case of a DMD patient, anoligonucleotide used will preferably correct one mutation as present inthe DMD gene of said patient and create a protein that will look like aBMD protein: said protein will preferably be a functional orsemi-functional dystrophin as later defined herein. In the case of a BMDpatient, an oligonucleotide as used will preferably correct one mutationas present in the BMD gene of said patient and create a dystrophin whichwill be more functional than the dystrophin which was originally presentin said BMD patient.

As defined herein, a functional dystrophin is preferably a wild typedystrophin corresponding to a protein having the amino acid sequence asidentified in SEQ ID NO: 1. As defined herein, a semi-functionaldystrophin is preferably a BMD-like dystrophin corresponding to aprotein having an acting binding domain in its N terminal part (first240 amino acids at the N terminus), a cysteine-rich domain (amino acid3361 till 3685) and a C terminal domain (last 325 amino acids at the Cterminus) each of these domains being present in a wild type dystrophinas known to the skilled person. The amino acids indicated hereincorrespond to amino acids of the wild type dystrophin being representedby SEQ ID NO:1. In other words, a functional or a semi-functionaldystrophin is a dystrophin which exhibits at least to some extent anactivity of a wild type dystrophin. “At least to some extent” preferablymeans at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%of a corresponding activity of a wild type functional dystrophin. Inthis context, an activity of a functional dystrophin is preferablybinding to actin and to the dystrophin-associated glycoprotein complex(DGC or DAPC) (Ehmsen J et al, 2002).

Binding of dystrophin to actin and to the DGC or DAPC complex may bevisualized by either co-immunoprecipitation using total protein extractsor immunofluorescence analysis of cross-sections using variousantibodies reacting with the different members of the complex, from acontrol (non-DMD) biopsy of one from a muscle suspected to bedystrophic, pre- and/or post-treatment, as known to the skilled person.

Individuals or patients suffering from Duchenne muscular dystrophytypically have a mutation in the gene encoding dystrophin (the DMD ordystrophin gene) that prevents synthesis of the complete protein, i.e. apremature stop codon prevents the synthesis of the C-terminus. In Beckermuscular dystrophy the dystrophin gene also comprises a mutationcompared to the wild-type but the mutation does typically not result ina premature stop codon and the C-terminus is typically synthesized. As aresult, a functional or semi-functional dystrophin protein issynthesized that has at least the same activity in kind as the wild typeprotein, although not necessarily the same amount of activity. Thegenome of a BMD patient typically encodes a dystrophin proteincomprising the N terminal part (first 240 amino acids at the Nterminus), a cysteine-rich domain (amino acid 3361 till 3685) and aC-terminal domain (last 325 amino acids at the C-terminus) but in themajority of cases its central rod shaped domain is shorter than the oneof a wild type dystrophin (Monaco et al., 1988). Antisenseoligonucleotide-induced exon skipping for the treatment of DMD istypically directed to overcome a premature stop in the pre-mRNA byskipping an exon, preferably in the central rod-domain shaped domain, tocorrect the open reading frame and allow synthesis of remainder of thedystrophin protein including the C-terminus, albeit that the protein issomewhat smaller as a result of a smaller rod domain. In a preferredembodiment, an individual having DMD and being treated by anoligonucleotide as defined herein will be provided a dystrophin whichexhibits at least to some extent an activity of a wild type dystrophin.More preferably, if said individual is a Duchenne patient or issuspected to be a Duchenne patient, a functional or a semi-functionaldystrophin is a dystrophin of an individual having BMD: typically saiddystrophin is able to interact with both actin and the DGC or DAPC, butits central rod shaped domain may be shorter than the one of a wild typedystrophin (Monaco et al., 1988). The central rod domain of wild typedystrophin comprises 24 spectrin-like repeats. For example, a centralrod shaped domain of a dystrophin as provided herein may comprise 5 to23, 10 to 22 or 12 to 18 spectrin-like repeats as long as it can bind toactin and to DGC.

Alleviating one or more symptom(s) of Duchenne Muscular Dystrophy orBecker Muscular Dystrophy in an individual using an oligonucleotide ofthe invention may be assessed by any of the following assays:prolongation of time to loss of walking, improvement of muscle strength,improvement of the ability to lift weight, improvement of the time takento rise from the floor, improvement in the nine-metre walking time,improvement in the time taken for four-stairs climbing, improvement ofthe leg function grade, improvement of the pulmonary function,improvement of cardiac function, improvement of the quality of life.Each of these assays is known to the skilled person. As an example, thepublication of Manzur et al (2008) gives an extensive explanation ofeach of these assays. For each of these assays, as soon as a detectableimprovement or prolongation of a parameter measured in an assay has beenfound, it will preferably mean that one or more symptoms of DuchenneMuscular Dystrophy or Becker Muscular Dystrophy has been alleviated inan individual using an oligonucleotide of the invention. Detectableimprovement or prolongation is preferably a statistically significantimprovement or prolongation as described in Hodgetts et al. (2006).Alternatively, the alleviation of one or more symptom(s) of DuchenneMuscular Dystrophy or Becker Muscular Dystrophy may be assessed bymeasuring an improvement of a muscle fiber function, integrity and/orsurvival. In a preferred method, one or more symptom(s) of a DMD or aBMD patient is/are alleviated and/or one or more characteristic(s) ofone or more muscle cells from a DMD or a BMD patient is/are improved.Such symptoms or characteristics may be assessed at the cellular, tissuelevel or on the patient self.

An alleviation of one or more characteristics of a muscle cell from apatient may be assessed by any of the following assays on a myogeniccell or muscle cell from a patient: reduced calcium uptake by musclecells, decreased collagen synthesis, altered morphology, altered lipidbiosynthesis, decreased oxidative stress, and/or improved muscle fiberfunction, integrity, and/or survival. These parameters are usuallyassessed using immunofluorescence and/or histochemical analyses of crosssections of muscle biopsies.

The improvement of muscle fiber function, integrity and/or survival maybe assessed using at least one of the following assays: a detectabledecrease of creatine kinase in blood, a detectable decrease of necrosisof muscle fibers in a biopsy cross-section of a muscle suspected to bedystrophic, and/or a detectable increase of the homogeneity of thediameter of muscle fibers in a biopsy cross-section of a musclesuspected to be dystrophic. Each of these assays is known to the skilledperson.

Creatine kinase may be detected in blood as described in Hodgetts et al.(2006). A detectable decrease in creatine kinase may mean a decrease of5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to theconcentration of creatine kinase in a same DMD or BMD patient beforetreatment.

A detectable decrease of necrosis of muscle fibers is preferablyassessed in a muscle biopsy, more preferably as described in Hodgetts etal. (2006), using biopsy cross-sections. A detectable decrease ofnecrosis may be a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or more of the area wherein necrosis has been identified usingbiopsy cross-sections. The decrease is measured by comparison to thenecrosis as assessed in a same DMD or BMD patient before treatment.

A detectable increase of the homogeneity of the diameter of a musclefiber is preferably assessed in a muscle biopsy cross-section, morepreferably as described in Hodgetts et al. (2006). The increase ismeasured by comparison to the homogeneity of the diameter of a musclefiber in a same DMD or BMD patient before treatment.

Preferably, an oligonucleotide of the invention provides said individualwith a functional or a semi-functional dystrophin protein (typically inthe case of DMD) and is able to, for at least in part decrease theproduction of an aberrant dystrophin protein in said individual(typically in the case of BMD).

Decreasing the production of an aberrant dystrophin mRNA, or aberrantdystrophin protein, preferably means that 90%, 80%, 70%, 60%, 50%, 40%,30%, 20%, 10%, 5% or less of the initial amount of aberrant dystrophinmRNA, or aberrant dystrophin protein, is still detectable by RT PCR(mRNA) or immunofluorescence or western blot analysis (protein). Anaberrant dystrophin mRNA or protein is also referred to herein as a lessfunctional (compared to a wild type functional dystrophin protein asearlier defined herein) or a non-functional dystrophin mRNA or protein.A non-functional dystrophin protein is preferably a dystrophin proteinwhich is not able to bind actin and/or members of the DGC proteincomplex. A non-functional dystrophin protein or dystrophin mRNA doestypically not have, or does not encode, a dystrophin protein with anintact C-terminus of the protein. The detection of a functional orsemi-functional dystrophin mRNA or protein may be done as for anaberrant dystrophin mRNA or protein.

Once a DMD patient is provided with a functional or a semi-functionaldystrophin protein, at least part of the cause of DMD is taken away.Hence, it would then be expected that the symptoms of DMD are at leastpartly alleviated. The enhanced skipping frequency also increases thelevel of functional or a semi-functional dystrophin protein produced ina muscle cell of a DMD or BMD individual.

Exons contain one or more specific sequences comprising splicingregulatory elements which have shown to be effective targets forantisense oligonucleotides (Aartsma-Rus et al, 2010). One embodimenttherefore provides an oligonucleotide for providing said individual witha functional or semi-functional dystrophin protein wherein saidoligonucleotide comprises a sequence which is specifically binding,targeting and/or hybridizing with and/or blocking these splicingregulatory elements in a dystrophin pre-mRNA exon. Such oligonucleotideis also able to bind and/or target and/or hybridize with and/or blockthese splicing regulatory elements in a dystrophin pre-mRNA. Inaddition, since an exon will only be included into the resulting mRNAwhen both the splice sites are recognized by the spliceosome complex,splice sites are other targets for an oligonucleotide of the invention.One embodiment therefore provides an oligonucleotide for providing saidindividual with a functional or semi-functional dystrophin proteinwherein said oligonucleotide comprises a sequence which is specificallybinding and/or targeting and/or hybridizing with, and/or blocking one ofor both the splice sites of an exon of a dystrophin pre-mRNA. Sucholigonucleotide is also able to bind and/or target, hybridize withand/or block one or both of these splice sites of an exon of adystrophin pre-mRNA. Usually, a splice site of an exon comprises 1, 2,3, or more nucleotides present in said exon and 1, 2, 3, or morenucleotides present in an adjacent or neighboring intron. In oneembodiment an oligonucleotide is used which is solely binding to and/ortargeting and/or hybridizing with an intron region of a dystrophinpre-mRNA. Such oligonucleotide is able to bind and/or able to targetand/or able to hybridize with said intron region. This is however notnecessary: it is also possible to use an oligonucleotide which targetsand/or binds and/or hybridizes with and/or is able to target and/or isable to binds and/or is able to hybridizes with an intron-specificsequence as well as exon-specific sequence. Of course, anoligonucleotide is not necessarily binding to and/or targeting and/orhybridizing with the entire sequence of a dystrophin exon or intron.Such oligonucleotide is also not necessarily able to bind to and/or ableto target and/or able to hybridize with the entire sequence of adystrophin exon or intron. Oligonucleotides which are specificallybinding, targeting and/or hybridizing with and/or which are specificallyable to bind and/or able to target and/or able to hybridize part of suchexon or intron are preferred. An oligonucleotide is used, saidoligonucleotide is preferably reverse complementary to, and/or binds to,and/or targets and/or hybridizes with and/or is able to bind to and/oris able to target and/or is able to hybridize with at least part of adystrophin exon and/or intron, said part having at least 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides.

Splicing of a dystrophin pre-mRNA occurs via two sequentialtransesterification reactions involving an intronic branch point and asplice site of an adjacent intron. Hence, an oligonucleotide is used forexon skipping, wherein said oligonucleotide comprises a sequence whichis binding to and/or targeting and/or hybridizing with or is able tobind to and/or is able to target and/or is able to hybridize with suchbranch point and/or splice site. Preferably said splice site and/orbranch point is present in a dystrophin pre-mRNA.

Since splice sites contain consensus sequences, the use of anoligonucleotide part or a functional equivalent thereof comprising asequence which is capable of binding to and/or able to bind to and/orable to target and/or able to hybridize and/or binds to and/or targetand/or hybridizes with a splice site involves the risk of promiscuoushybridization. Hybridization of said oligonucleotide to other splicesites than the sites of the exon to be skipped could easily interferewith the accuracy of the splicing process. To overcome these and otherpotential problems related to the use of an oligonucleotide which isbinding and/or hybridizing and/or targeting and/or is able to bind toand/or is able to target and/or is able to hybridize a splice site, mostpreferred embodiment provides an oligonucleotide for providing saidindividual with a functional or a semi-functional dystrophin protein,wherein said oligonucleotide or a functional equivalent thereof, bindingto and/or hybridizing with and/or targeting and/or is able to bind toand/or is able to hybridize and/or is able to target a specific part ofa dystrophin pre-mRNA exon. Exons contain coding sequences which aretypically more specific that the non-coding intron sequences.Preferably, said oligonucleotide binding to and/or hybridizing withand/or targeting and/or able to bind to and/or able to hybridize withand/or able to target a specific part of a dystrophin pre-mRNA exon iscapable of specifically blocking, interfering and/or inhibiting asplicing regulatory sequence and/or structure of the anticipated exon(s)in said dystrophin pre-mRNA. Interfering with such splicing regulatorysequence and/or structure has the advantage that such elements arelocated within the exon. The risk for sequence-related off-targeteffects is therefore limited. By providing an oligonucleotide for theinterior of the exon to be skipped, it is possible to mask the exon fromthe splicing apparatus. The failure of the splicing apparatus torecognize the exon to be skipped thus leads to exclusion of the exonfrom the final mRNA. This embodiment does not interfere directly withthe enzymatic process of the splicing machinery (the joining of theexons). It is thought that this allows the method to be more specificand/or reliable. It has been found that an oligonucleotide capable ofbinding to and/or able to bind to and/or able to target and/or able tohybridize and/or binding to and/or hybridizing with and/or targeting anexon at any point may be able to induce the skipping of said exon.

Within the context of the invention, an oligonucleotide of the inventionmay comprise a functional equivalent or an equivalent of anoligonucleotide. A functional equivalent or an equivalent of anoligonucleotide preferably means an oligonucleotide as defined hereinwherein one or more nucleotides have been substituted and wherein anactivity of said functional equivalent or equivalent is retained to atleast some extent. Preferably, an activity of said oligonucleotidecomprising a functional equivalent or equivalent of an oligonucleotideis providing a functional or a semi-functional dystrophin protein. Saidactivity of said oligonucleotide comprising a functional equivalent oran equivalent of an oligonucleotide is therefore preferably assessed byquantifying the amount of a functional or a semi-functional dystrophinprotein. A functional or semi-functional dystrophin is herein preferablydefined as being a dystrophin able to bind actin and members of the DGC(or DAPC) protein complex. The assessment of said activity of saidfunctional equivalent of an oligonucleotide is preferably done by RT-PCRand sequencing (on RNA level; for detection of specific exon skipping),or by immunofluorescence and Western blot analyses (on protein level:for detection of protein restoration). Said activity is preferablyretained to at least some extent when it represents at least 50%, or atleast 60%, or at least 70% or at least 80% or at least 90% or at least95% or more of corresponding activity of said oligonucleotide thefunctional equivalent or equivalent derives from. Throughout thisapplication, when the word oligonucleotide is used it may be replaced bya functional equivalent thereof or an equivalent thereof as definedherein. In an embodiment, an equivalent or a functional equivalent of anoligonucleotide of the invention comprises a modification. Throughoutthis application, when the word oligonucleotide is used it may bereplaced by an antisense oligonucleotide as defined herein unlessotherwise indicated.

Hence, the use of an oligonucleotide or a functional equivalent thereof,or an equivalent thereof comprising a 2′-O-methyl phosphorothioate RNAmonomer or consisting of 2′-O-methyl phosphorothioate RNA and comprisinga 5-methylpyrimidine (i.e. a 5-methylcytosine and/or a 5-methyluracil)and/or a 2,6-diaminopurine base and being represented by a nucleotidesequence comprising or consisting of a sequence which is reversecomplementary to, and/or binds to and/or targets and/or hybridizesand/or is able to bind to and/or is able to target and/or is able tohybridize with a dystrophin pre-mRNA exon is assumed to have a positiveeffect on at least one of the parameters of said oligonucleotide, as hasalready been defined herein, when compared to their counterparts whichdo not comprise any 5-methylcytosine, 5-methyluracil and2,6-diaminopurine (i.e. so called non-modified oligonucleotide) asindicated earlier herein, and is therefore assumed to exhibit animproved therapeutic result in a DMD or a BMD cell of a patient and/orin a DMD or a BMD patient. Such a therapeutic result may becharacterized by:

-   -   alleviating one or more symptom(s) of DMD or BMD and/or    -   alleviating one or more characteristics of a muscle cell from a        patient and/or    -   providing said individual with a functional or semi-functional        dystrophin protein and/or    -   at least in part decreasing the production of an aberrant        dystrophin protein in said individual.

Each of these features has already been defined herein.

Preferably, an oligonucleotide is represented by a nucleotide sequencewhich comprises or consists of a sequence which is binding to and/ortargeting and/or being reverse complementary to and/or is hybridizingwith and/or which is able to bind to and/or is able to target and/or isable to hybridize with and/or is reverse complementary to at least apart of dystrophin pre-mRNA exons 44 to 55, said oligonucleotide havinga length of at least 10 nucleotides. However, the length of saidoligonucleotide may be at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides.Throughout the invention, said sequence representing the oligonucleotidemay also be called a base or a nucleotide sequence.

Preferably, an oligonucleotide of the invention is represented by anucleotide sequence or a base sequence comprising or consisting of asequence that is capable of binding to, and/or targeting and/or beingreverse complementary to and/or hybridizing with and/or being able tobind to and/or being able to hybridize with and/or being able to targeta part of an exon of dystrophin pre-mRNA. Said binding or targeted partmay be at least 50% of the length of the oligonucleotide of theinvention, or at least 60%, or at least 70%, or at least 80%, or atleast 90% or at least 95%, or 98% and up to 100%. An oligonucleotide maybe represented by a nucleotide or a base sequence, said nucleotide orbase sequence comprising a sequence that binds and/or targets and/or isreverse complementary to and/or hybridizes with and/or is able to bindto and/or is able to hybridize with and/or is able to target at least apart of an exon selected from the group consisting of exons 44 to 55 ofdystrophin pre-mRNA as defined herein and additional flanking sequences.In a more preferred embodiment, the length of said binding or targetedpart of said oligonucleotide is of at least 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides. Several types of flanking sequences may be used.Preferably, flanking sequences are used to modify the binding of aprotein to said oligonucleotide, or to modify a thermodynamic propertyof said oligonucleotide, more preferably to modify target RNA bindingaffinity. In another preferred embodiment, additional flanking sequencesare reverse complementary to sequences of the dystrophin pre-mRNA whichare not present in said exon. Such flanking sequences are preferablycapable of binding to and/or targeting sequences comprising orconsisting of the branchpoint and/or the splice site acceptor or donorconsensus sequences of said exon. In a preferred embodiment, suchflanking sequences are capable of binding to and/or targeting sequencescomprising or consisting of sequences of an intron of the dystrophinpre-mRNA which is adjacent to said exon.

One preferred embodiment provides an oligonucleotide for providing saidindividual with a functional or a semi-functional dystrophin protein,said oligonucleotide or a functional equivalent thereof or an equivalentthereof, being represented by a sequence or a base sequence whichcomprises:

-   -   a sequence which binds, is able to bind, targets, hybridizes or        is reverse complementary to a region of a dystrophin pre-mRNA        exon that is hybridized to another part of a dystrophin pre-mRNA        exon (closed structure), and    -   a sequence which binds and/or targets and/or hybridizes and/or        is reverse complementary to and/or is able to bind and/or is        able to target and/or is able to hybridize with a region of a        dystrophin pre-mRNA exon that is not hybridized in said        dystrophin pre-mRNA (open structure).

For this embodiment, reference is made to the WO 2004/083446 patentapplication. RNA molecules exhibit strong secondary structures, mostlydue to base pairing of complementary or partly complementary stretcheswithin the same RNA. It has long since been thought that structures inthe RNA play a role in the function of the RNA. Without being bound bytheory, it is believed that the secondary structure of the RNA of anexon plays a role in structuring the splicing process. Through itsstructure, an exon is recognized as a part that needs to be included inthe mRNA. In an embodiment, an oligonucleotide is capable of interferingwith the structure of the exon and therefore capable of interfering withthe splicing apparatus of said exon, masking the exon from the splicingapparatus and thereby inducing the skipping of said exon. It has beenfound that many oligonucleotides indeed comprise this capacity, somemore efficient than others. Without being bound by theory it is thoughtthat the overlap with an open structure improves the invasion efficiencyof the oligonucleotide (i.e., increases the efficiency with which theoligonucleotide can enter the structure), whereas the overlap with theclosed structure subsequently increases the efficiency of interferingwith the secondary structure of the RNA of the exon. It is found thatthe length of the partial reverse complementarity to both the closed andthe open structure is not extremely restricted. We have observed highefficiencies with compounds comprising oligonucleotides with variablelengths of reverse complementarity in either structure. The term(reverse) complementarity is used herein to refer to a stretch ofnucleic acids that can hybridise to another stretch of nucleic acidsunder physiological conditions. Hybridization conditions are laterdefined herein. It is thus not absolutely required that all the bases inthe region of complementarity are capable of pairing with bases in theopposing strand. For instance, when designing an antisenseoligonucleotide, one may want to incorporate for instance a residue thatdoes not base pair with the base on the complementary strand. Mismatchesmay to some extent be allowed, if under the circumstances in the cell,the stretch of nucleotides is capable of hybridizing to thecomplementary part.

In a preferred embodiment a reverse complementary part of an antisenseoligonucleotide (either to said open or to said closed structure)comprises at least 3, and more preferably at least 4 consecutivenucleotides. The reverse complementary regions are preferably designedsuch that, when combined, they are specific for an exon in a pre-mRNA.Such specificity may be created with various lengths of reversecomplementary regions as this depends on the actual sequences in other(pre-)mRNA in the system. The risk that also one or more other pre-mRNAwill be able to hybridise to an oligonucleotide decreases withincreasing size of said oligonucleotide. It is clear that an antisenseoligonucleotide comprising mismatches in the region of reversecomplementarity but that retain the capacity to hybridise to thetargeted region(s) in the pre-mRNA, can be used in the presentinvention. However, preferably at least the reverse complementary partsdo not comprise such mismatches as these typically have a higherefficiency and a higher specificity than oligonucleotide having suchmismatches in one or more reverse complementary regions. It is thoughtthat higher hybridisation strengths, (i.e. increasing number ofinteractions with the opposing strand) are favourable in increasing theefficiency of the process of interfering with the splicing machinery ofthe system. Preferably, the reverse complementarity is from 90 to 100%.In general this allows for 1 or 2 mismatch(es) in an oligonucleotide of20 nucleotides or 1 to 4 mismatches in an oligonucleotide of 40nucleotides. Therefore, we may have 1, 2, 3, 4, 5 mismatches in anoligonucleotide of 10 to 50 nucleotides. Preferably, 0, 1 or 2mismatches are present in an oligonucleotide of 10 to 50 nucleotides.

The structure (i.e., open and closed structures) is best analyzed in thecontext of the pre-mRNA wherein the exon resides. Such structure may beanalyzed in the actual RNA. However, it is currently possible to predictthe secondary structure of an RNA molecule (at lowest energy costs)quite well using structure-modeling programs. Non-limiting examples of asuitable program are RNA structure version 4.5 or RNA mfold version 3.5(Zuker et al., 2003). A person skilled in the art will be able topredict, with suitable reproducibility, a likely structure of an exon,given a nucleotide sequence. Best predictions are obtained whenproviding such modeling programs with both said exon and flanking intronsequences. It is typically not necessary to model the structure of theentire pre-mRNA.

The open and closed structure to which the oligonucleotide of anoligonucleotide is directed, are preferably adjacent to one another. Itis thought that in this way the annealing of the oligonucleotide to theopen structure induces opening of the closed structure whereuponannealing progresses into this closed structure. Through this action thepreviously closed structure assumes a different conformation. However,when potential (cryptic) splice acceptor and/or donor sequences arepresent within the targeted exon, occasionally a new exon inclusionsignal or splicing regulatory sequence, element, structure, or signal isgenerated defining a different (neo) exon, i.e., with a different 5′end, a different 3′ end, or both. This type of activity is within thescope of the present invention as the targeted exon is excluded from themRNA. The presence of a new exon, containing part of the targeted exon,in the mRNA does not alter the fact that the targeted exon, as such, isexcluded. The inclusion of a neo-exon can be seen as a side effect whichoccurs only occasionally. There are two possibilities when exon skippingis used to restore (part of) an open reading frame of dystrophin that isdisrupted as a result of a mutation. One is that the neo-exon isfunctional in the restoration of the reading frame, whereas in the othercase the reading frame is not restored. When selecting a compoundcomprising an oligonucleotide for restoring dystrophin reading frames bymeans of exon-skipping it is of course clear that under these conditionsonly those compounds comprising those oligonucleotides are selected thatindeed result in exon-skipping that restores the dystrophin open readingframe, with or without a neo-exon.

Further provided is an oligonucleotide for providing said individualwith a functional or a semi-functional dystrophin protein, wherein saidoligonucleotide or a functional equivalent thereof or an equivalentthereof comprises a 2′-O-methyl phosphorothioate RNA monomer or consistsof 2′-O-methyl phosphorothioate RNA and comprises a 5-methylpyrimidine(i.e. a 5-methylcytosine, and/or a 5-methyluracil) and/or a2,6-diaminopurine base and is represented by a nucleotide or a basesequence comprising a sequence that is reverse complementary to and/orbinds to and/or targets and/or hybridizes with and/or is able to bind toand/or is able to target and/or is able to hybridize with a binding sitefor a serine-arginine (SR) protein in RNA of an exon of a dystrophinpre-mRNA. In WO 2006/112705 patent application we have disclosed thepresence of a correlation between the effectivity of an exon-internalantisense oligonucleotide in inducing exon skipping and the presence ofa (for example by ESEfinder) predicted SR binding site in the targetpre-mRNA site of said AON. Therefore, in one embodiment anoligonucleotide is generated comprising determining a (putative) bindingsite for an SR (Ser-Arg) protein in RNA of a dystrophin exon andproducing a corresponding compound comprising oligonucleotide that isreverse complementary to and/or binds to and/or targets and/orhybridizes with and/or is able to bind and/or is able to target and/oris able to hybridize with said RNA and that at least partly overlapssaid (putative) binding site. The term “at least partly overlaps” isdefined herein as to comprise an overlap of only a single nucleotide ofan SR binding site as well as multiple nucleotides of said binding siteas well as a complete overlap of said binding site. This embodimentpreferably further comprises determining from a secondary structure ofsaid RNA, a region that is hybridized to another part of said RNA(closed structure) and a region that is not hybridized in said structure(open structure), and subsequently generating an oligonucleotide that atleast partly overlaps said (putative) binding site and that overlaps atleast part of said closed structure and overlaps at least part of saidopen structure. In this way we increase the chance of obtaining anoligonucleotide that is capable of interfering with the exon inclusionfrom the pre-mRNA into mRNA. It is possible that a first selectedSR-binding region does not have the requested open-closed structure inwhich case another (second) SR protein binding site is selected which isthen subsequently tested for the presence of an open-closed structure.This process is continued until a sequence is identified which containsan SR protein binding site as well as a(n) (partly overlapping)open-closed structure. This sequence is then used to design anoligonucleotide which is reverse complementary to said sequence.

Such a method for generating an antisense oligonucleotide is alsoperformed by reversing the described order, i.e. first generating anoligonucleotide comprising determining, from a secondary structure ofRNA from a dystrophin exon, a region that assumes a structure that ishybridised to another part of said RNA (closed structure) and a regionthat is not hybridised in said structure (open structure), andsubsequently generating an oligonucleotide, of which at least a part ofsaid oligonucleotide is reverse complementary to said closed structureand of which at least another part of said oligonucleotide is reversecomplementary to said open structure. This is then followed bydetermining whether an SR protein binding site at least overlaps withsaid open/closed structure. In this way the method of WO 2004/083446 isimproved. In yet another embodiment the selections are performedsimultaneously.

Without wishing to be bound by any theory it is currently thought thatuse of an oligonucleotide directed to or targeting an SR protein bindingsite results in (at least partly) impairing the binding of an SR proteinto the binding site of an SR protein which results in disrupted orimpaired splicing.

Preferably, an open/closed structure and an SR protein binding sitepartly overlap and even more preferred an open/closed structurecompletely overlaps an SR protein binding site or an SR protein bindingsite completely overlaps an open/closed structure. This allows for animproved disruption of exon inclusion.

Besides consensus splice site and branchpoint intronic sequences, many(if not all) exons contain splicing regulatory sequences such as but notlimited to exonic splicing enhancer (ESE) sequences to facilitate therecognition of genuine splice sites by the spliceosome (Cartegni et al.,2002; and Cartegni et al., 2003). A subgroup of splicing factors, calledthe SR proteins, can bind to these ESEs and recruit other splicingfactors, such as U1 and U2AF to (weakly defined) splice sites. Thebinding sites of the four most abundant SR proteins (SF2/ASF, SC35,SRp40 and SRp55) have been analyzed in detail and these results areimplemented in ESEfinder, a web source that predicts potential bindingsites for these SR proteins (Cartegni et al., 2002; and Cartegni et al.,2003). There is a correlation between the effectiveness of anoligonucleotide and the presence/absence of an SF2/ASF, SC35 and SRp40binding site in the site targeted by said oligonucleotide. In apreferred embodiment, the invention thus provides an oligonucleotide asdescribed above, which is reverse complementary to and/or targets and/orbinds to and/or hybridizes with and/or is able to target and/or is ableto bind and/or is able to hybridize with a binding site for a SRprotein. Preferably, said SR protein is SF2/ASF or SC35 or SRp40.

In one embodiment a DMD patient is provided with a functional or asemi-functional dystrophin protein by using an oligonucleotide or afunctional equivalent thereof or an equivalent thereof comprising a2′-O-methyl phosphorothioate RNA monomer or consisting of 2′-O-methylphosphorothioate RNA and comprising a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase and being capable of specifically binding or targeting and/or beingable to bind and/or being able to target and/or being able to hybridizea regulatory RNA sequence which is required for the correct splicing ofa dystrophin exon in a transcript. Several cis-acting RNA sequences arerequired for the correct splicing of exons in a transcript. Inparticular, elements such as an exonic splicing enhancer (ESE), an exonrecognition sequence (ERS), and/or an exonic splicing silencer (ESS) areidentified to regulate specific and efficient splicing of constitutiveand alternative exons. Using a sequence-specific antisenseoligonucleotide or a base-specific antisense oligonucleotide (AON) thatbinds to and/or targets and/or is reverse complementary to and/orhybridizes with and/or is able to bind and/or is able to hybridize withand/or is able to target the elements, their regulatory function isdisturbed so that the exon is skipped, as shown for DMD. Hence, in onepreferred embodiment, an oligonucleotide or a functional equivalentthereof or an equivalent thereof is used which is reverse complementaryto and/or binds to and/or targets and/or hybridizes with and/or is ableto bind to and/or is able to target and/or is able to hybridize with anexonic splicing enhancer (ESE), an exon recognition sequence (ERS),and/or an exonic splicing silencer (ESS).

In a preferred embodiment, an oligonucleotide of the invention comprisesor consists of a sequence or a base sequence that is reversecomplementary to and/or binds to and/or targets and/or hybridizes withand/or is able to bind to and/or is able to target and/or is able tohybridize with at least a part of dystrophin pre-mRNA exon 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, or 55, said part having at least 10nucleotides. However, said part may also have at least 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,or 33 nucleotides. For the dystrophin exons identified above, we providethe stretch of nucleotides (SEQ ID NO: 2 to 13 identified below) of saidexon to which an oligonucleotide binds to and/or is reversecomplementary to and/or targets and/or hybridizes with and/or is able tobind to and/or is able to target and/or is able to hybridize with.

(SEQ ID NO: 2) 5′- GCGAUUUGACAGAUCUGUUGAGAAAUGGCGGCGUUUUCAUUAUGAUAUAAAGAUAUUUAAUCAGUGGCUAACAGAAGCUGAACAGUUUCUCAGAAAGACACAAAUUCCUGAGAAUUGGGAACAUGCUAAAUACAAAUGGUAUCUUAAG-3′ for skipping of exon 44; (SEQ ID NO: 3) 5′-GAACUCCAGGAUGGCAUUGGGCAGCGGCAAACUGUUGUCAGAACAUUGAAUGCAACUGGGGAAGAAAUAAUUCAGCAAUCCUCAAAAACAGAUGCCAGUAUUCUACAGGAAAAAUUGGGAAGCCUGAAUCUGCGGUGGCAGGAGGUCUGCAAACAGCUGUCAGACAGAAAAAAGAG-3′ for skipping of exon 45; (SEQ ID NO: 4)5′- GCUAGAAGAACAAAAGAAUAUCUUGUCAGAAUUUCAAAGAGAUUUAAAUGAAUUUGUUUUAUGGUUGGAGGAAGCAGAUAACAUUGCUAGUAUCCCACUUGAACCUGGAAAAGAGCAGCAACUAAAAGAAAAGCUUGAGCAAGUCAAG-3′ for skipping of exon 46; (SEQ ID NO: 5) 5′-UUACUGGUGGAAGAGUUGCCCCUGCGCCAGGGAAUUCUCAAACAAUUAAAUGAAACUGGAGGACCCGUGCUUGUAAGUGCUCCCAUAAGCCCAGAAGAGCAAGAUAAACUUGAAAAUAAGCUCAAGCAGACAAAUCUCCAGUGGAUAAAG-3′ for skipping of exon 47; (SEQ ID NO: 6) 5′-GUUUCCAGAGCUUUACCUGAGAAACAAGGAGAAAUUGAAGCUCAAAUAAAAGACCUUGGGCAGCUUGAAAAAAAGCUUGAAGACCUUGAAGAGCAGUUAAAUCAUCUGCUGCUGUGGUUAUCUCCUAUUAGGAAUCAGUUGGAAAUUUAUAACCAACCAAACCAAGAAGGACCAUUUGACGUUCAG-3′ for skipping of exon 48;(SEQ ID NO: 7) 5′- GAAACUGAAAUAGCAGUUCAAGCUAAACAACCGGAUGUGGAAGAGAUUUUGUCUAAAGGGCAGCAUUUGUACAAGGAAAAACCAGCCACUCAGCCAGUGAAG-3′ for skipping of exon 49; (SEQ ID NO: 8) 5′-AGGAAGUUAGAAGAUCUGAGCUCUGAGUGGAAGGCGGUAAACCGUUUACUUCAAGAGCUGAGGGCAAAGCAGCCUGACCUAGCUCCUGGACUGACCACUAUUGGAGCCU-3′ for skipping of exon 50; (SEQ ID NO: 9) 5′-CUCCUACUCAGACUGUUACUCUGGUGACACAACCUGUGGUUACUAAGGAAACUGCCAUCUCCAAACUAGAAAUGCCAUCUUCCUUGAUGUUGGAGGUACCUGCUCUGGCAGAUUUCAACCGGGCUUGGACAGAACUUACCGACUGGCUUUCUCUGCUUGAUCAAGUUAUAAAAUCACAGAGGGUGAUGGUGGGUGACCUUGAGGAUAUCAACGAGAUGAUCAUCAAGCAGAAG-3′ for skipping of exon 51;(SEQ ID NO: 10) 5′- GCAACAAUGCAGGAUUUGGAACAGAGGCGUCCCCAGUUGGAAGAACUCAUUACCGCUGCCCAAAAUUUGAAAAACAAGACCAGCAAUCAAGAGGCUAGAACAAUCAUUACGGAUCGAA-3′ for skipping of exon 52; (SEQ ID NO: 11) 5′-UUGAAAGAAUUCAGAAUCAGUGGGAUGAAGUACAAGAACACCUUCAGAACCGGAGGCAACAGUUGAAUGAAAUGUUAAAGGAUUCAACACAAUGGCUGGAAGCUAAGGAAGAAGCUGAGCAGGUCUUAGGACAGGCCAGAGCCAAGCUUGAGUCAUGGAAGGAGGGUCCCUAUACAGUAGAUGCAAUCCAAAAGAAAAUCACAGAAACCAAG-3′ for skipping of exon 53; (SEQ ID NO: 12) 5′-CAGUUGGCCAAAGACCUCCGCCAGUGGCAGACAAAUGUAGAUGUGGCAAAUGACUUGGCCCUGAAACUUCUCCGGGAUUAUUCUGCAGAUGAUACCAGAAAAGUCCACAUGAUAACAGAGAAUAUCAAUGCCUCUUGGAGAAGCAUUCAUAAAAG-3′ for skipping of exon 54; (SEQ ID NO: 13) 5′-GGUGAGUGAGCGAGAGGCUGCUUUGGAAGAAACUCAUAGAUUACUGCAACAGUUCCCCCUGGACCUGGAAAAGUUUCUUGCCUGGCUUACAGAAGCUGAAACAACUGCCAAUGUCCUACAGGAUGCUACCCGUAAGGAAAGGCUCCUAGAAGACUCCAAGGGAGUAAAAGAGCUGAUGAAACAAUGGCAA-3′ for skipping of exon 55.

Therefore, a preferred oligonucleotide comprises a2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase and binds to and/or is reverse complementary to and/or targetsand/or hybridizes with and/or is able to bind and/or is able to targetand/or is able to hybridize with a continuous stretch of at least 10 andup to 33 nucleotides within one of the following exon nucleotidesequences selected from SEQ ID NO: 2 to 13.

Preferred oligonucleotides are also defined as follows:

-   -   comprise a 2′-O-methyl phosphorothioate RNA monomer or consist        of 2′-O-methyl phosphorothioate RNA and    -   bind to and/or are reverse complementary to and/or target and/or        hybridize with and/or is able to bind to and/or is able to        target and/or is able to hybridize with a continuous stretch of        at least 10 and up to 33 nucleotides within one of the following        exon nucleotide sequences selected from SEQ ID NO: 2 to 13 as        identified above.

More preferably, such oligonucleotides comprise a 5-methylpyrimidine(i.e., a 5-methylcytosine, and/or a 5-methyluracil) and/or a2,6-diaminopurine base as earlier defined herein.

More preferred oligonucleotides comprise a 2′-O-methyl phosphorothioateRNA monomer or consist of 2′-O-methyl phosphorothioate RNA and morepreferably comprise a 5-methylpyrimidine (i.e., a 5-methylcytosine,and/or a 5-methyluracil) and/or a 2,6-diaminopurine base and arerepresented by a nucleotide or a base sequence comprising or consistingof SEQ ID NO: 14-90 or by a nucleotide or a base sequence comprising orconsisting of a fragment of SEQ ID NO: 14-90. SEQ ID NO:14-90 areidentified in Table 1.

In this context, “a 5-methylpyrimidine” means at least one5-methylpyrimidine. Accordingly, “at least one 5-methylpyrimidine” meansat least one 5-methylcytosine and/or at least one 5-methyluracil.

Accordingly, preferred non-modified oligonucleotides are preferablyderived from one of the nucleotide or base sequences SEQ ID NO:14-90with X=C, Y=U, Z=A), and/or are represented by SEQ ID NO:91, 93, 94-170.Each of these non-modified oligonucleotides comprises no5-methylpyrimidine (i.e., a 5-methylcytosine, and/or a 5-methyluracil)and no 2,6-diaminopurine. Please note that SEQ ID NO:91 is identicalwith SEQ ID NO: 132.

Accordingly, preferred modified oligonucleotides are derived from one ofthe nucleotide or base sequences SEQ ID NO:14-90 and comprise at leastone 5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a5-methyluracil) and/or at least one 2,6-diaminopurine (i.e. at least oneX is m⁵C=X₁ and/or at least one Y is m⁵U=Y₁ and/or at least one Z isa²A=Z₁). Please note that SEQ ID NO: 92 is identical with SEQ ID NO:199. More preferred modified oligonucleotides are represented by anucleotide or a base sequence comprising or consisting of SEQ ID NO: 92,171-213, 215, 217, 218, 219. Even more preferred modifiedoligonucleotides (all X=m⁵C=X₁ and/or all Y=m⁵U=Y₁ and/or all Z=a²A=Z₁)are derived from the most preferred nucleotide or base sequences (SEQ IDNO:15, 21, 31, 40, 52, and 57) and are represented by SEQ ID NO: 92,171-174, 185-188, 199, 200, 202-215, 217, 218, 219. The most preferredmodified oligonucleotides are disclosed in Table 3.

Within the context of the invention, a fragment of SEQ ID NO:14-90, or afragment of SEQ ID NO:91-219, preferably means a nucleotide or a basesequence comprising or consisting of at least 10 contiguous nucleotidesfrom said SEQ ID NO:14-90 or from said SEQ ID NO:91-219.

Such more preferred oligonucleotides are also defined as follows:

-   -   comprise a 2′-O-methyl phosphorothioate RNA monomer or consist        of 2′-O-methyl phosphorothioate RNA and    -   are represented by a nucleotide or base sequence comprising or        consisting of SEQ ID NO: 14-90, 91, 93-170 or by a nucleotide or        base sequence comprising or consisting of a fragment of SEQ ID        NO: 14-90, 91, 93-170.

More preferably, such oligonucleotides comprise a 5-methylpyrimidine(i.e., a 5-methylcytosine, and/or a 5-methyluracil) and/or a2,6-diaminopurine base as earlier defined herein.

Even more preferred oligonucleotides comprise a 2′-O-methylphosphorothioate RNA monomer or consist of 2′-O-methyl phosphorothioateRNA and more preferably comprise a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, are represented by a nucleotide or a base sequence comprising orconsisting of SEQ ID NO: 14-90, 92, 171-215, 217, 218, 219 or by anucleotide or a base sequence comprising or consisting of a fragment ofSEQ ID NO:14-90, 92, 171-215, 217, 218, 219 and having a length of 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32 or 33 nucleotides. Preferred sequences (i.e. preferrednucleotide or base sequences) among SEQ ID NO:14-90, 92, and 171-215,217, 218, 219 include SEQ ID NO: 15, 21, 31, 40, 43, 52, 57, 59,171-174, 185-188, 199, 200, 202-213, 215, 217, 218, 219 more preferablySEQ ID NO: 40, 43, 52, 57, 59, 208, 207, 200, 210, 206, 171, 173, 199,213, 185, 187.

Such even more preferred oligonucleotides are also defined as follows:

-   -   comprise a 2′-O-methyl phosphorothioate RNA monomer or consist        of 2′-O-methyl phosphorothioate RNA and        are represented by a nucleotide or a base sequence comprising or        consisting of SEQ ID NO: 14-90, 91, 93-170, 216 or by a        nucleotide or a base sequence comprising or consisting of a        fragment of SEQ ID NO: 14-90, 91, 93-170 and have a length of        10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,        26, 27, 28, 29, 30, 31, 32 or 33 nucleotides. More preferably,        such oligonucleotides comprise a 5-methylpyrimidine (i.e. a        5-methylcytosine, and/or a 5-methyluracil) and/or a        2,6-diaminopurine base as earlier defined herein.

Even more preferably, such modified oligonucleotides are represented bya nucleotide or a base sequence comprising or consisting of SEQ ID NO:92, 171-213, 215 217, 218, 219 or by a nucleotide or a base sequencecomprising or consisting of a fragment of SEQ ID NO: 92, 171-213, 215,217, 218, 219 and have a length of 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides. Even more preferred modified oligonucleotides are derivedfrom the most preferred nucleotide or base sequences (SEQ ID NO:15, 21,31, 40, 52, and 57) and are represented by a nucleotide or a basesequence comprising or consisting of SEQ ID NO: 92, 171-174, 185-188,199, 200, 202-213, 215, 217, 218, 219 or by a nucleotide or a basesequence comprising or consisting of a fragment of SEQ ID NO: 92,171-174, 185-188, 199, 200, 202-213, 215, 217, 218, 219 and having alength of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32 or 33 nucleotides.

Preferred oligonucleotides for inducing the skipping of exon 44 from thedystrophin pre-mRNA are as follows below.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 14 and has a length of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:14comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:14.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:14 isrepresented by SEQ ID NO:94 and a preferred fragment of SEQ ID NO:94 isrepresented by SEQ ID NO:143.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA and is represented by a nucleotide or a basesequence comprising SEQ ID NO: 94 and has a length of 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by afragment of SEQ ID NO:94 comprising or consisting of at least 10contiguous nucleotides or bases of SEQ ID NO:94.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides. A preferred fragment of SEQ ID NO:14 comprises SEQ ID NO:63 and a preferred fragment of SEQ ID NO:94 comprises SEQ ID NO: 143,and each of said preferred fragments has a length of 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 15 and has a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:15comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:15.

Accordingly a non-modified oligonucleotide derived from SEQ ID NO:15 isrepresented by SEQ ID NO:95.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or base sequencecomprising SEQ ID NO: 95 and has a length of 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of SEQ IDNO:95 comprising or consisting of at least 10 contiguous nucleotides orbases of SEQ ID NO:95.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides. A preferred fragment of SEQ ID NO:15 comprises SEQ ID NO:64 and a preferred fragment of SEQ ID NO:95 comprises SEQ ID NO:144 andeach of said fragments has a length of 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 nucleotides. Accordingly,more preferably, said oligonucleotide comprises a 5-methylpyrimidine(i.e. a 5-methylcytosine, and/or a 5-methyluracil) and/or a2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

Such preferred oligonucleotide is also defined as follows:

-   -   comprises a 2′-O-methyl phosphorothioate RNA monomer or consists        of 2′-O-methyl phosphorothioate RNA and    -   is represented by a nucleotide or base sequence comprising or        consisting of SEQ ID NO: 15 or 95 or 64 or 144 and has a length        of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33        nucleotides or by a nucleotide or base sequence comprising or        consisting of a fragment of SEQ ID NO: 15 or 95 or 64 or 144,        said fragment comprising or consisting of at least 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32 or 33 contiguous nucleotides or bases of SEQ ID        NO:15 or 95 or 64 or 144.

More preferably, such oligonucleotide comprises a 5-methylpyrimidine(i.e. a 5-methylcytosine, and/or a 5-methyluracil) and/or a2,6-diaminopurine base as earlier defined herein

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or base        sequence comprising SEQ ID NO: 15 and has a length of 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or        by a fragment of SEQ ID NO:15 comprising or consisting of at        least 10 contiguous nucleotides or bases of SEQ ID NO:15. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or base        sequence comprising SEQ ID NO: 204 and has a length of 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or        by a fragment of SEQ ID NO:204 comprising or consisting of at        least 10 contiguous nucleotides or bases of SEQ ID NO:204. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or base        sequence comprising SEQ ID NO: 208 and has a length of 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or        by a fragment of SEQ ID NO:208 comprising or consisting of at        least 10 contiguous nucleotides or bases of SEQ ID NO:208. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its uraciles have been replaced by 5-methyluraciles and all        its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or base        sequence comprising SEQ ID NO: 205 and has a length of 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or        by a fragment of SEQ ID NO:205 comprising or consisting of at        least 10 contiguous nucleotides or bases of SEQ ID NO:205. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its adenines have been replaced by 2,6-diaminopurines,    -   such oligonucleotide is represented by a nucleotide or base        sequence comprising SEQ ID NO: 207 and has a length of 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or        by a fragment of SEQ ID NO:207 comprising or consisting of at        least 10 contiguous nucleotides or bases of SEQ ID NO:207. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or base sequence comprising SEQ IDNO: 16 and has a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:16 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:16.

Accordingly, anon-modified oligonucleotide derived from SEQ ID NO:16 isrepresented by SEQ ID NO:96.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 96 and has a length of 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of SEQ IDNO:96 comprising or consisting of at least 10 contiguous nucleotides orbases of SEQ ID NO:96.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 17 and has a length of 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or33 nucleotides or by a fragment of SEQ ID NO:17 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:17.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:17 isrepresented by SEQ ID NO:97 and a preferred fragment of SEQ ID NO:97 isrepresented by SEQ ID NO:145.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 97 and has a length of 23, 24, 25, 26, 27, 28, 29,30, 31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:97comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:97.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides. A preferred fragment of SEQ ID NO:17 comprises SEQ ID NO:65 and a preferred fragment of SEQ ID NO: 97 comprises SEQ ID NO: 145,each of said fragments has a length of 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 18 and has a length of 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or33 nucleotides or by a fragment of SEQ ID NO:18 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:18.

Accordingly, anon-modified oligonucleotide derived from SEQ ID NO:18 isrepresented by SEQ ID NO:98 and a preferred fragment of SEQ ID NO:98 isrepresented by SEQ ID NO:146.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides. A preferred fragment of SEQ ID NO:18 comprises SEQ ID NO:66 and a preferred fragment of SEQ ID NO: 98 comprises SEQ ID NO: 146,each of said fragments has a length of 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 nucleotides.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 98 and has a length of 23, 24, 25, 26, 27, 28, 29,30, 31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:98comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:98.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 19 and has a length of 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:19 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:19.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:19 isrepresented by SEQ ID NO:99.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 99 and has a length of 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:99comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:99.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 20 and has a length of 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or33 nucleotides or by a fragment of SEQ ID NO:20 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:20.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:20 isrepresented by SEQ ID NO:100 and a preferred fragment of SEQ ID NO:100is represented by SEQ ID NO:147, 148 or 149.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 100 and has a length of 23, 24, 25, 26, 27, 28,29, 30, 31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:100comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:100.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides. A preferred fragment of SEQ ID NO:20 comprises SEQ ID NO:67 and a preferred fragment of SEQ ID NO:100 comprises SEQ ID NO:147,each of said fragments has a length of 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32 or 33 nucleotides. Another preferred fragment of SEQID NO:20 comprises SEQ ID NO: 68 and another preferred fragment of SEQID NO:100 comprises SEQ ID NO: 148, each of said fragments has a lengthof 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32 or 33 nucleotides. Another preferred fragment of SEQ ID NO:20comprises SEQ ID NO: 69 and another preferred fragment of SEQ ID NO:100comprises SEQ ID NO: 149, each of said fragments has a length of 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32 or 33 nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

Preferred oligonucleotides for inducing the skipping of exon 45 from thedystrophin pre-mRNA are as follows below.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 21 and has a length of 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides or by a fragment of SEQ ID NO:21 comprising or consisting ofat least 10 contiguous nucleotides or bases of SEQ ID NO:21.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:21 isrepresented by SEQ ID NO:101 and a preferred fragment of SEQ ID NO:101is represented by SEQ ID NO:150, 151 or 152.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 101 and has a length 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides or by a fragment of SEQ ID NO:101 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:101.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides. A preferred fragment of SEQ ID NO:21 comprises SEQ ID NO:70 and a preferred fragment of SEQ ID NO:101 comprises SEQ ID NO:150,each of said fragments has a length of 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32 or 33 nucleotides. Another preferred fragment of SEQID NO:21 comprises SEQ ID NO: 71 and another preferred fragment of SEQID NO:101 comprises SEQ ID NO:151, each of said fragments has a lengthof 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 nucleotides.Another preferred fragment of SEQ ID NO:21 comprises SEQ ID NO: 72 and apreferred fragment of SEQ ID NO:101 comprises SEQ ID NO:152, each ofsaid fragments has a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32 or 33 nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

Such preferred oligonucleotide is also defined as follows:

-   -   comprises a 2′-O-methyl phosphorothioate RNA monomer or consists        of 2′-O-methyl phosphorothioate RNA and    -   is represented by a nucleotide or a base sequence comprising or        consisting of SEQ ID NO: 21 and has a length of 25, 26, 27, 28,        29, 30, 31, 32, or 33 nucleotides or by a nucleotide or a base        sequence comprising or consisting of a fragment of SEQ ID NO:        21, said fragment comprising or consisting of at least 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32 or 33 contiguous nucleotides or bases of SEQ        ID NO:21.

More preferably, such oligonucleotide comprises a 5-methylpyrimidine(i.e. a 5-methylcytosine, and/or a 5-methyluracil) and/or a2,6-diaminopurine base as earlier defined herein.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 21 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of        SEQ ID NO:21 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:21.

Accordingly, said oligonucleotide is particularly represented by anucleotide or a base sequence comprising SEQ ID NO: 200 and has a lengthof 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment ofSEQ ID NO:200 comprising or consisting of at least 10 contiguousnucleotides or bases of SEQ ID NO:200.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its uraciles have been replaced by 5-methyluraciles and all        its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 21 or SEQ ID NO:209 in        particular, and has a length of 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, or 33 nucleotides, or by a fragment of SEQ        ID NO:21 or 209 comprising or consisting of at least 10        contiguous nucleotides or bases of SEQ ID NO: 21 or 209. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its adenines have been replaced by 2,6-diaminopurines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 21 or SEQ ID NO: 210 in        particular, and has a length of 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, or 33 nucleotides, or by a fragment of SEQ        ID NO:21 or 210 comprising or consisting of at least 10        contiguous nucleotides or bases of SEQ ID NO:21 or 210. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 22 and has a length of 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides or by a fragment of SEQ ID NO:22 comprising or consisting ofat least 10 contiguous nucleotides or bases of SEQ ID NO:22.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:22 isrepresented by SEQ ID NO:102.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 102 and has a length 24, 25, 26, 27, 28, 29, 30,31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:102 comprisingor consisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:102.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 23 and has a length of 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides or by a fragment of SEQ ID NO:23 comprising or consisting ofat least 10 contiguous nucleotides or bases of SEQ ID NO:23.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:23 isrepresented by SEQ ID NO:103.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or base sequencecomprising SEQ ID NO: 103 and has a length 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides or by a fragment of SEQ ID NO:103 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:103.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 24 and has a length of 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or33 nucleotides or by a fragment of SEQ ID NO:24 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:24.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:24 isrepresented by SEQ ID NO:104.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or base sequencecomprising SEQ ID NO: 104 and has a length 23, 24, 25, 26, 27, 28, 29,30, 31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:104comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:104.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 25 and has a length of 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides or by a fragment of SEQ ID NO:25 comprising or consisting ofat least 10 contiguous nucleotides or bases of SEQ ID NO:25.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:25 isrepresented by SEQ ID NO:105.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 105 and has a length 24, 25, 26, 27, 28, 29, 30,31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:105 comprisingor consisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:105.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 26 and has a length of 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides or by a fragment of SEQ ID NO:26 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:26.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:26 isrepresented by SEQ ID NO:106.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 106 and has a length 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:106comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:106.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 27 and has a length of 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides or by a fragment of SEQ ID NO:27 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:27.

Accordingly a non-modified oligonucleotide derived from SEQ ID NO:27 isrepresented by SEQ ID NO:107.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 107 and has a length 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:107comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:107.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 28 and has a length of 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides or by a fragment of SEQ ID NO:28 comprising or consisting ofat least 10 contiguous nucleotides or bases of SEQ ID NO:28.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:28 isrepresented by SEQ ID NO:108. Each of SEQ ID NO:28 and SEQ ID NO:108identified in table 1 comprises a hypoxanthine base at position 7.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 108 and has a length 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides or by a fragment of SEQ ID NO:108 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:108.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 29 and has a length of 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides or by a fragment of SEQ ID NO:29 comprising or consisting ofat least 10 contiguous nucleotides or bases of SEQ ID NO:29.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:29 isrepresented by SEQ ID NO:109.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 109 and has a length 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides or by a fragment of SEQ ID NO:109 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:109.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 30 and has a length of 30, 31, 32 or 33 nucleotides or by afragment of SEQ ID NO:30 comprising or consisting of at least 10contiguous nucleotides or bases of SEQ ID NO:30.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:30 isrepresented by SEQ ID NO:110.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 110 and has a length 30, 31, 32, or 33 nucleotidesor by a fragment of SEQ ID NO:110 comprising or consisting of at least10 contiguous nucleotides or bases of SEQ ID NO:110.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

Preferred oligonucleotides for inducing the skipping of exon 51 from thedystrophin pre-mRNA are as follows below.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 31 and has a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:31comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:31.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:31 isrepresented by SEQ ID NO:111 and a preferred fragment of SEQ ID NO:111is represented by SEQ ID NO:153 or 154.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 111 and has a length 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of SEQ IDNO:111 comprising or consisting of at least 10 contiguous nucleotides orbases of SEQ ID NO:111.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides. A preferred fragment of SEQ ID NO:31 comprises SEQ ID NO:73 and a preferred fragment of SEQ ID NO: 111 comprises SEQ ID NO: 153,and each of said fragments has a length of 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 nucleotides.Another preferred fragment of SEQ ID NO:31 comprises SEQ ID NO: 74 andanother preferred fragment of SEQ ID NO: 111 comprises SEQ ID NO: 154,and each of said fragments has a length of 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

Such preferred oligonucleotide is also defined as follows:

-   -   comprises a 2′-O-methyl phosphorothioate RNA monomer or consists        of 2′-O-methyl phosphorothioate RNA and    -   is represented by a nucleotide or a base sequence comprising or        consisting of SEQ ID NO: 31 and has a length of 20, 21, 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a        nucleotide or a base sequence comprising or consisting of a        fragment of SEQ ID NO: 31, said fragment comprising or        consisting of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        contiguous nucleotides or bases of SEQ ID NO:31.

More preferably, such oligonucleotide comprises a 5-methylpyrimidine(i.e. a 5-methylcytosine, and/or a 5-methyluracil) and/or a2,6-diaminopurine base as earlier defined herein.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 31 or SEQ ID NO: 215 and has a        length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or        33 nucleotides, or by a fragment of SEQ ID NO:31 or SEQ ID        NO:215 comprising or consisting of at least 10 contiguous        nucleotides of SEQ ID NO:31 or of SEQ ID NO: 215. Such fragment        has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18,        19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 202 and has a length of 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides,        or by a fragment of SEQ ID NO:202 comprising or consisting of at        least 10 contiguous nucleotides or bases of SEQ ID NO:202. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines and        all its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 203 and has a length of 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides,        or by a fragment of SEQ ID NO:203 comprising or consisting of at        least 10 contiguous nucleotides or bases of SEQ ID NO:203. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its adenines have been replaced by 2,6-diaminopurines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 206 and has a length of 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides,        or by a fragment of SEQ ID NO:206 comprising or consisting of at        least 10 contiguous nucleotides or bases of SEQ ID NO:206. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 32 and has a length of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a fragment of SEQ IDNO:32 comprising or consisting of at least 10 contiguous nucleotides orbases of SEQ ID NO:32.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:32 isrepresented by SEQ ID NO:112.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 112 and has a length 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragmentof SEQ ID NO:112 comprising or consisting of at least 10 contiguousnucleotides or bases of SEQ ID NO:112.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 33 and has a length of 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides or by a fragment of SEQ ID NO:33 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:33.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:33 isrepresented by SEQ ID NO:113.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 113 and has a length 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:113comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:113.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In another embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and is represented by a nucleotide or a base sequence comprising SEQID NO: 34 and has a length of 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides or by a fragment of SEQ ID NO:34 comprising or consisting ofat least 10 contiguous nucleotides or bases of SEQ ID NO:34.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:34 isrepresented by SEQ ID NO:114.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consist of 2′-O-methylphosphorothioate RNA is represented by a nucleotide sequence comprisingSEQ ID NO: 114 and has a length 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides or by a fragment of SEQ ID NO:114 comprising or consistingof at least 10 contiguous nucleotides of SEQ ID NO:114.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides. A preferred fragment of SEQ ID NO: 34 comprises or consistsof SEQ ID NO: 93 (PS1116: 5′-CAACAUCAAGGAAGAUGGCAUUUCU-3′).

Such preferred oligonucleotide is also defined as follows:

-   -   comprises a 2′-O-methyl phosphorothioate RNA monomer or consists        of 2′-O-methyl phosphorothioate RNA and    -   is represented by a nucleotide or a base sequence comprising or        consisting of SEQ ID NO: 34 or 93 or 114 and has a length of 25,        26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a nucleotide        sequence comprising or consisting of a fragment of SEQ ID NO: 34        or 93 or 114, said fragment comprising or consisting of at least        10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,        26, 27, 28, 29, 30, 31, 32 or 33 contiguous nucleotides or bases        of SEQ ID NO:34 or 93 or 114.

More preferably, such oligonucleotide comprises a 5-methylpyrimidine(i.e. a 5-methylcytosine, and/or a 5-methyluracil) and/or a2,6-diaminopurine base as earlier defined herein

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 34 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a fragment of        SEQ ID NO:34 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:34. Such fragment has        preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its adenines have been replaced by 2,6-diaminopurines    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 34 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a fragment of        SEQ ID NO:34 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:34. Such fragment has        preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 35 and has a length of 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or33 nucleotides or by a fragment of SEQ ID NO:35 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:35.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:35 isrepresented by SEQ ID NO:115.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 115 and has a length 23, 24, 25, 26, 27, 28, 29,30, 31, 32, or 33 nucleotides, or by a fragment of SEQ ID NO:115comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:115.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 36 and has a length of 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or33 nucleotides or by a fragment of SEQ ID NO:36 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:36.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:36 isrepresented by SEQ ID NO:116 and a preferred fragment of SEQ ID NO:116is represented by SEQ ID NO:155 or 156 or 157.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 116 and has a length 23, 24, 25, 26, 27, 28, 29,30, 31, 32, or 33 nucleotides, or by a fragment of SEQ ID NO:116comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:116.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides. A preferred fragment of SEQ ID NO:36 comprises SEQ ID NO:75 or a preferred fragment of SEQ ID NO: 116 comprises SEQ ID NO: 155,and each of said fragments has a length of 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides. Another preferred fragment of SEQ ID NO:36 comprises SEQ IDNO: 76 or another preferred fragment of SEQ ID NO: 116 comprises SEQ IDNO: 156, and each of said fragments has a length of 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides. Another preferred fragment of SEQ ID NO:36 comprises SEQ IDNO: 77 or another preferred fragment of SEQ ID NO: 116 comprises SEQ IDNO: 157, and each of said fragments has a length of 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32or 33 nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 37 and has a length of 30, 31, 32 or 33 nucleotides or by afragment of SEQ ID NO:37 comprising or consisting of at least 10contiguous nucleotides or bases of SEQ ID NO:37.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:37 isrepresented by SEQ ID NO:117.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 117 and has a length 30, 31, 32, or 33 nucleotidesor by a fragment of SEQ ID NO:117 comprising or consisting of at least10 contiguous nucleotides or bases of SEQ ID NO:117.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 38 and has a length of 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides or by a fragment of SEQ ID NO:38 comprising or consisting ofat least 10 contiguous nucleotides or bases of SEQ ID NO:38.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:38 isrepresented by SEQ ID NO:118.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 118 and has a length 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides, or by a fragment of SEQ ID NO:118 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:118.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

Preferred oligonucleotides for inducing the skipping of exon 52 from thedystrophin pre-mRNA are as follows below.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 39 and has a length of 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides or by a fragment of SEQ ID NO:39 comprising or consisting ofat least 10 contiguous nucleotides or bases of SEQ ID NO:39.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:39 isrepresented by SEQ ID NO:119.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 119 and has a length 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides or by a fragment of SEQ ID NO:119 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:119.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 201 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of        SEQ ID NO:201 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:201. Such fragment has        preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 40 and has a length of 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides, or by a fragment of SEQ ID NO:40 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:40. Such fragment has preferably a length of 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly a non-modified oligonucleotide derived from SEQ ID NO:40 isrepresented by SEQ ID NO:120 and a preferred fragment of SEQ ID NO:120is represented by SEQ ID NO:158 or 159 or 160.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 120 and has a length 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:120comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:120.

A preferred fragment of SEQ ID NO:40 comprises SEQ ID NO: 78 and apreferred fragment of SEQ ID NO:120 comprises SEQ ID NO:158, and eachfragment has a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32 or 33 nucleotides. Another preferred fragment ofSEQ ID NO:40 comprises SEQ ID NO: 79 and another preferred fragment ofSEQ ID NO:120 comprises SEQ ID NO:159, and each fragment has a length of13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32 or 33 nucleotides. Another preferred fragment of SEQ ID NO:40comprises SEQ ID NO: 80 and another preferred fragment of SEQ ID NO:120comprises SEQ ID NO:160, and each fragment has a length of 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32 or 33 nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

Such preferred oligonucleotide is also defined as follows:

-   -   comprises a 2′-O-methyl phosphorothioate RNA monomer or consists        of 2′-O-methyl phosphorothioate RNA and    -   is represented by a nucleotide or a base sequence comprising or        consisting of SEQ ID NO: 40 or 120 and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a        nucleotide sequence comprising or consisting of a fragment of        SEQ ID NO: 40 or 120, said fragment comprising or consisting of        at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 contiguous nucleotides        or bases of SEQ ID NO:40 or 120.

More preferably, such oligonucleotide comprises a 5-methylpyrimidine(i.e. a 5-methylcytosine, and/or a 5-methyluracil) and/or a2,6-diaminopurine base as earlier defined herein

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 40 and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a        fragment of SEQ ID NO:40 comprising or consisting of at least 10        contiguous nucleotides or bases of SEQ ID NO:40. Accordingly,        said oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 171 and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a        fragment of SEQ ID NO:171 comprising or consisting of at least        10 contiguous nucleotides or bases of SEQ ID NO:171. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides. It is also encompassed that not all the 4        cytosines of SEQ ID NO:40 are modified as represented in SEQ ID        NO:171. It is encompassed that 1, 2 or 3 of these cytosines are        modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO:172 and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a        fragment of SEQ ID NO:172 comprising or consisting of at least        10 contiguous nucleotides or bases of SEQ ID NO:172. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides. It is also encompassed that not all the 7        uraciles of SEQ ID NO:40 are modified as represented in SEQ ID        NO:172. It is encompassed that 1, 2, 3, 4, 5 or 6 of these        uraciles are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its adenines have been replaced by 2,6-diaminopurines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 173 and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a        fragment of SEQ ID NO:173 comprising or consisting of at least        10 contiguous nucleotides or bases of SEQ ID NO:173. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides. It is also encompassed that not all the 5        adenines of SEQ ID NO:40 are modified as represented in SEQ ID        NO:173. It is encompassed that 1, 2, 3 or 4 of these adenines        are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines and        all its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 174 and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a        fragment of SEQ ID NO:174 comprising or consisting of at least        10 contiguous nucleotides or bases of SEQ ID NO:174.        Accordingly, said oligonucleotide is represented by a nucleotide        or a base sequence comprising SEQ ID NO: 174 and has a length of        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides,        or by a fragment of SEQ ID NO:174 comprising or consisting of at        least 10 contiguous nucleotides or bases of SEQ ID NO:174. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides. It is also encompassed that not all the 4        cytosines and not all the 7 uraciles of SEQ ID NO:40 are        modified as represented in SEQ ID NO:174. It is encompassed that        1, 2 or 3 of these cytosines and-or 1, 2, 3, 4, 5 or 6 of these        uraciles are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines and        all its adenines have been replaced by 2,6-diaminopurines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 175 and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a        fragment of SEQ ID NO:175 comprising or consisting of at least        10 contiguous nucleotides or bases of SEQ ID NO:175.        Accordingly, said oligonucleotide is represented by a nucleotide        sequence comprising SEQ ID NO: 175 and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a        fragment of SEQ ID NO:175 comprising or consisting of at least        10 contiguous nucleotides or bases of SEQ ID NO:175. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides. It is also encompassed that not all the 4        cytosines and not all the 5 adenines of SEQ ID NO:40 are        modified as represented in SEQ ID NO:175. It is encompassed that        1, 2 or 3 of these cytosines and-or 1, 2, 3 or 4 of these        adenines are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its adenines have been replaced by 2,6-diaminopurines and        all its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 176 and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a        fragment of SEQ ID NO:176 comprising or consisting of at least        10 contiguous nucleotides or bases of SEQ ID NO:176. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides. It is also encompassed that not all the 5        adenines and not all the 7 uraciles of SEQ ID NO: 40 are        modified as represented in SEQ ID NO:176. It is encompassed that        1, 2, 3 or 4 of these adenines and-or 1, 2, 3, 4, 5 or 6 of        these uraciles are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its adenines have been replaced by 2,6-diaminopurines, all        its cytosines have been replaced by 5-methylcytosines and all        its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 177 and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a        fragment of SEQ ID NO:177 comprising or consisting of at least        10 contiguous nucleotides or bases of SEQ ID NO:177. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides. It is also encompassed that not all the 4        cytosines and not all the 7 uraciles and not all the 5 adenines        of SEQ ID NO:40 are modified as represented in SEQ ID NO:177. It        is encompassed that 1, 2 or 3 of these cytosines and-or 1, 2, 3,        4, 5 or 6 of these uraciles and-or 1, 2, 3 or 4 of these        adenines are modified.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide sequence or a base comprising SEQID NO: 41 and has a length of 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides, or by a fragment of SEQ ID NO:41 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:41.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:41 isrepresented by SEQ ID NO:121.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 121 and has a length 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides, or by a fragment of SEQ ID NO:121 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:121.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 42 and has a length of 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides or by a fragment of SEQ ID NO:42 comprising or consisting ofat least 10 contiguous nucleotides or bases of SEQ ID NO:42.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:42 isrepresented by SEQ ID NO:122.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 122 and has a length 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides, or by a fragment of SEQ ID NO:122 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:122.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 43 and has a length of 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides, or by a fragment of SEQ ID NO:43 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:43.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Such preferred oligonucleotide is also defined as follows:

-   -   comprises a 2′-O-methyl phosphorothioate RNA monomer or consists        of 2′-O-methyl phosphorothioate RNA and    -   is represented by a nucleotide or a base sequence comprising or        consisting of SEQ ID NO: 43 or 123 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides or by a nucleotide        sequence comprising or consisting of a fragment of SEQ ID NO: 43        or 123, said fragment comprising or consisting of at least 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32 or 33 contiguous nucleotides or bases of        SEQ ID NO:43 or 123. Accordingly a non-modified oligonucleotide        derived from SEQ ID NO:43 is represented by SEQ ID NO:123 and a        preferred fragment of SEQ ID NO:123 is represented by SEQ ID NO:        161.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 123 and has a length 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, or 33 nucleotides, or by a fragment of SEQ ID NO:123comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:123.

More preferably, such oligonucleotide comprises a 5-methylpyrimidine(i.e. a 5-methylcytosine, and/or a 5-methyluracil) and/or a2,6-diaminopurine base as earlier defined herein. Even more preferably,said oligonucleotide has all its cytosines and/or all its uracil and/orall its adenines that have been substituted or modified as definedherein.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 43 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of        SEQ ID NO:43 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:43. Such fragment has        preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides. Accordingly, said oligonucleotide is represented by        a nucleotide or a base sequence comprising SEQ ID NO: 178 and        has a length of 25, 26, 27, 28, 29, 30, 31, 32, or 33        nucleotides, or by a fragment of SEQ ID NO:178 comprising or        consisting of at least 10 contiguous nucleotides or bases of SEQ        ID NO:178. It is also encompassed that not all the 6 cytosines        of SEQ ID NO:43 are modified as represented in SEQ ID NO:178. It        is encompassed that 1, 2, 3, 4 or 5 of these cytosines are        modified.

A preferred fragment of SEQ ID NO:43 comprises SEQ ID NO: 81 and apreferred fragment of SEQ ID NO:123 comprises SEQ ID NO:161, each ofsaid fragments has a length of10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 nucleotides.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO:179 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a fragment of        SEQ ID NO:179 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:179. Such fragment has        preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides. It is also encompassed that not all the 11 uraciles        of SEQ ID NO:43 are modified as represented in SEQ ID NO:179. It        is encompassed that 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of these        uraciles are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its adenines have been replaced by 2,6-diaminopurines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 180 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of        SEQ ID NO:180 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:180. Such fragment has        preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides. It is also encompassed that not all the 2 adenines        of SEQ ID NO:43 are modified as represented in SEQ ID NO:180.

It is encompassed that 1 of these adenines are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines and        all its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 181 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of        SEQ ID NO:181 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:181. Accordingly, said        oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 181 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of        SEQ ID NO:181 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:181. Such fragment has        preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides. It is also encompassed that not all the 6 cytosines        and not all the 11 uraciles of SEQ ID NO: 43 are modified as        represented in SEQ ID NO:181. It is encompassed that 1, 2, 3, 4        or 5 of these cytosines and-or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10        of these uraciles are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines and        all its adenines have been replaced by 2,6-diaminopurines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 182 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of        SEQ ID NO:182 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:182. Accordingly, said        oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 182 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of        SEQ ID NO:182 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:182. Such fragment has        preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides. It is also encompassed that not all the 6 cytosines        and not all the 2 adenines of SEQ ID NO:43 are modified as        represented in SEQ ID NO:182. It is encompassed that 1, 2, 3, 4        or 5 of these cytosines and-or 1 of these adenines are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its adenines have been replaced by 2,6-diaminopurines and        all its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 183 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of        SEQ ID NO:183 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:183. Such fragment has        preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides. It is also encompassed that not all the 2 adenines        and not all the 11 uraciles of SEQ ID NO:43 are modified as        represented in SEQ ID NO:183. It is encompassed that 1 of these        adenines and/or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of these        uraciles are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its adenines have been replaced by 2,6-diaminopurines, all        its cytosines have been replaced by 5-methylcytosines and all        its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 184 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of        SEQ ID NO:184 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:184. Such fragment has        preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides. It is also encompassed that not all the 6 cytosines        and not all the 11 uraciles and not all the 2 adenines of SEQ ID        NO:43 are modified as represented in SEQ ID NO:184. It is        encompassed that 1, 2, 3, 4 or 5 of these cytosines and-or 1, 2,        3, 4, 5, 6, 7, 8, 9 or 10 of these uraciles and-or 1 of these        adenines are modified.

Preferred oligonucleotides for inducing the skipping of exon 53 from thedystrophin pre-mRNA are as follows below.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 44 and has a length of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:44comprising or consisting of at least 10 contiguous or bases nucleotidesof SEQ ID NO:44.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:44 isrepresented by SEQ ID NO:124.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 124 and has a length 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a fragment ofSEQ ID NO:124 comprising or consisting of at least 10 contiguousnucleotides or bases of SEQ ID NO:124.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 45 and has a length of 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides, or by a fragment of SEQ ID NO:45 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:45.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:45 isrepresented by SEQ ID NO:125.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 125 and has a length 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:125comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:125.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 46 and has a length of 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides, or by a fragment of SEQ ID NO:46 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:46.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:46 isrepresented by SEQ ID NO:126.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 126 and has a length 24, 25, 26, 27, 28, 29, 30,31, 32, or 33 nucleotides or by a fragment of SEQ ID NO:126 comprisingor consisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:126.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 47 and has a length of 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides, or by a fragment of SEQ ID NO:47 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:47.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:47 isrepresented by SEQ ID NO:127.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 127 and has a length 24, 25, 26, 27, 28, 29, 30,31, 32, or 33 nucleotides, or by a fragment of SEQ ID NO:127 comprisingor consisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:127.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 48 and has a length of 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides or by a fragment of SEQ ID NO:48 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:48.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:48 isrepresented by SEQ ID NO:128.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 128 and has a length 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, or 33 nucleotides, or by a fragment of SEQ ID NO:128comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:128.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 49 and has a length of 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or33 nucleotides, or by a fragment of SEQ ID NO:49 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:49.

Accordingly a non-modified oligonucleotide derived from SEQ ID NO:49 isrepresented by SEQ ID NO:129.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 129 and has a length 23, 24, 25, 26, 27, 28, 29,30, 31, 32, or 33 nucleotides, or by a fragment of SEQ ID NO:129comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:129.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 50 and has a length of 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides, or by a fragment of SEQ ID NO:50 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:50.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:50 isrepresented by SEQ ID NO:130.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 130 and has a length 24, 25, 26, 27, 28, 29, 30,31, 32, or 33 nucleotides, or by a fragment of SEQ ID NO:130 comprisingor consisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:130.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 51 and has a length of 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides, or by a fragment of SEQ ID NO:51 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:51.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:51 isrepresented by SEQ ID NO:131.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 131 and has a length 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides, or by a fragment of SEQ ID NO:131 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:131.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 52 and has a length of 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides, or by a fragment of SEQ ID NO:52 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:52.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:52 isrepresented by SEQ ID NO: 91 and a preferred fragment of SEQ ID NO:91 isrepresented by SEQ ID NO:162, 163 or 164. SEQ ID NO: 91 is identicalwith SEQ ID NO: 132.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 91 and has a length 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides, or by a fragment of SEQ ID NO:191 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:91.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

Such preferred oligonucleotide is also defined as follows:

-   -   comprises a 2′-O-methyl phosphorothioate RNA monomer or consists        of 2′-O-methyl phosphorothioate RNA and    -   is represented by a nucleotide or base sequence comprising or        consisting of SEQ ID NO: 52 or 91 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides or by a nucleotide        sequence comprising or consisting of a fragment of SEQ ID NO: 52        or 91, said fragment comprising or consisting of at least 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32 or 33 contiguous nucleotides or bases of        SEQ ID NO:52 or 91.

More preferably, such oligonucleotide comprises a 5-methylpyrimidine(i.e. a 5-methylcytosine, and/or a 5-methyluracil) and/or a2,6-diaminopurine base as earlier defined herein.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or base        sequence comprising SEQ ID NO: 52 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of        SEQ ID NO:52 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:52. Such fragment has        preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides.

A preferred fragment of SEQ ID NO:52 comprises SEQ ID NO: 82 and apreferred fragment of SEQ ID NO:91 comprises SEQ ID NO:162, each of saidfragments has a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32 or 33 nucleotides. Another preferred fragment of SEQ ID NO:52comprises SEQ ID NO: 83 and another preferred fragment of SEQ ID NO:91comprises SEQ ID NO:163, each of said fragments has a length of 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 nucleotides. Anotherpreferred fragment of SEQ ID NO:52 comprises SEQ ID NO: 84 and anotherpreferred fragment of SEQ ID NO:91 comprises SEQ ID NO:164, each of saidfragments has a length of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32 or 33 nucleotides. A most preferred fragment of SEQID NO: 52 comprises or consists of SEQ ID NO: 91 (PS229L:5′-GUUGCCUCCGGUUCUGAAGGUGUUC-3′). Another most preferred fragment of SEQID NO: 52 comprises or consists of SEQ ID NO: 92 (PS524:5′-GUUGXXUXXGGUUXUGAAGGUGUUX-3′; wherein X is 5-methylcytosine).

Such preferred oligonucleotide is also defined as follows:

-   -   comprises a 2′-O-methyl phosphorothioate RNA monomer or consists        of 2′-O-methyl phosphorothioate RNA and    -   is represented by a nucleotide or a base sequence comprising or        consisting of SEQ ID NO: 82, 83, 84, 91 or 92 or 162 or 163 or        164 and has a length of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides or by a nucleotide or        a base sequence comprising or consisting of a fragment of SEQ ID        NO: 82, 83, 84, 91 or 92, or 162 or 163 or 164, said fragment        comprising or consisting of at least 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 contiguous nucleotides or bases of SEQ ID NO:82, 83, 84,        91, or 92 or 162, 163 or 164.

More preferably, such oligonucleotide comprises a 5-methylpyrimidine(i.e. a 5-methylcytosine, and/or a 5-methyluracil) and/or a2,6-diaminopurine base as earlier defined herein

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 82, 83, 84 or 92 and has a length        of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,        32, or 33 nucleotides, or by a fragment of SEQ ID NO:82, 83, 84,        or 92 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:82, 83, 84, or 92. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides. SEQ ID NO: 92 is identical with SEQ ID        NO: 199. It is also encompassed that not all the 6 cytosines of        SEQ ID NO:52 are modified as represented in SEQ ID NO:92. It is        encompassed that 1, 2, 3, 4 or 5 of these cytosines are        modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   two of its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 218 and has a length of 17, 18,        19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33        nucleotides, or by a fragment of SEQ ID NO:218 comprising or        consisting of at least 10 contiguous nucleotides or bases of SEQ        ID NO:218. Such fragment has preferably a length of 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32 or 33 nucleotides.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   three of its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 219 and has a length of 17, 18,        19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33        nucleotides, or by a fragment of SEQ ID NO:219 comprising or        consisting of at least 10 contiguous nucleotides or bases of SEQ        ID NO:219. Such fragment has preferably a length of 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32 or 33 nucleotides.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   four of its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 217 and has a length of 17, 18,        19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33        nucleotides, or by a fragment of SEQ ID NO:217 comprising or        consisting of at least 10 contiguous nucleotides or bases of SEQ        ID NO:217. Such fragment has preferably a length of 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32 or 33 nucleotides.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 211 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a fragment of        SEQ ID NO:211 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:211. Accordingly, said        oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 211 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a fragment of        SEQ ID NO:211 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:211. Such fragment has        preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides. It is also encompassed that not all the 9 uraciles        of SEQ ID NO:52 are modified as represented in SEQ ID NO:211. It        is encompassed that 1, 2, 3, 4, 5, 6, 7, or 8 of these uraciles        are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines and        all its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 212 and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a        fragment of SEQ ID NO:212 comprising or consisting of at least        10 contiguous nucleotides or bases of SEQ ID NO:212.        Accordingly, said oligonucleotide is represented by a nucleotide        or a base sequence comprising SEQ ID NO: 212 and has a length of        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides,        or by a fragment of SEQ ID NO:212 comprising or consisting of at        least 10 contiguous nucleotides or bases of SEQ ID NO:212. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides. It is also encompassed that not all the 6        cytosines and not all the 9 uraciles of SEQ ID NO:52 are        modified as represented in SEQ ID NO:212. It is encompassed that        1, 2, 3, 4, or 5 of these cytosines and/or 1, 2, 3, 4, 5, 6, 7,        or 8 of these uraciles are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its adenines have been replaced by 2,6-diaminopurines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 213 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a fragment of        SEQ ID NO:213 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:213. Such fragment has        preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides. It is also encompassed that not all the 2 adenines        of SEQ ID NO:52 are modified as represented in SEQ ID NO:213. It        is encompassed that 1 of these adenines are modified.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 53 and has a length of 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides, or by a fragment of SEQ ID NO:53 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:53.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:53 isrepresented by SEQ ID NO:133.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 133 and has a length 25, 26, 27, 28, 29, 30, 31,32, or 33 nucleotides, or by a fragment of SEQ ID NO:133 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:133.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 54 and has a length of 30, 31, 32 or 33 nucleotides, or by afragment of SEQ ID NO:54 comprising or consisting of at least 10contiguous nucleotides or bases of SEQ ID NO:54.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:54 isrepresented by SEQ ID NO:134.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 134 and has a length 30, 31, 32, or 33nucleotides, or by a fragment of SEQ ID NO:134 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:134.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 55 and has a length of 30, 31, 32 or 33 nucleotides, or by afragment of SEQ ID NO:55 comprising or consisting of at least 10contiguous nucleotides or bases of SEQ ID NO:55.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:55 isrepresented by SEQ ID NO:135.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 135 and has a length 30, 31, 32, or 33nucleotides, or by a fragment of SEQ ID NO:135 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:135.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 56 and has a length of 33, 34 or 35 nucleotides or by a fragmentof SEQ ID NO:56 comprising or consisting of at least 10 contiguousnucleotides or bases of SEQ ID NO:56.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:56 isrepresented by SEQ ID NO:136.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 136 and has a length 33, 34 or 35 nucleotides, orby a fragment of SEQ ID NO:136 comprising or consisting of at least 10contiguous nucleotides or bases of SEQ ID NO:136.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

Preferred oligonucleotides for inducing the skipping of exon 55 from thedystrophin pre-mRNA are as follows below.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 57 and has a length of 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides or by a fragment of SEQ ID NO:57 comprising or consisting ofat least 10 contiguous nucleotides or bases of SEQ ID NO:57. Suchfragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Such preferred oligonucleotide is also defined as follows:

-   -   comprises a 2′-O-methyl phosphorothioate RNA monomer or consists        of 2′-O-methyl phosphorothioate RNA and    -   is represented by a nucleotide or a base sequence comprising or        consisting of SEQ ID NO: 57 and has a length of 24, 25, 26, 27,        28, 29, 30, 31, 32, or 33 nucleotides or by a nucleotide        sequence comprising or consisting of a fragment of SEQ ID NO:        57, said fragment comprising or consisting of at least 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32 or 33 contiguous nucleotides or bases of SEQ        ID NO:57.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:57 isrepresented by SEQ ID NO:137 and a preferred fragment of SEQ ID NO:137is represented by SEQ ID NO:165 or 166.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA and is represented by a nucleotide or a basesequence comprising SEQ ID NO: 137 and has a length 24, 25, 26, 27, 28,29, 30, 31, 32, or 33 nucleotides, or by a fragment of SEQ ID NO:137comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:137.

More preferably, such oligonucleotide comprises a 5-methylpyrimidine(i.e. a 5-methylcytosine, and/or a 5-methyluracil) and/or a2,6-diaminopurine base as earlier defined herein. Even more preferably,said oligonucleotide has all its cytosines and/or all its uracil and/orall its adenines that have been substituted or modified as definedherein.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 57 and has a length of 24, 25,        26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment        of SEQ ID NO:57 comprising or consisting of at least 10        contiguous nucleotides or bases of SEQ ID NO:57.

Accordingly, said oligonucleotide is represented by a nucleotide or abase sequence comprising SEQ ID NO: 185 and has a length of 24, 25, 26,27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of SEQ IDNO:185 comprising or consisting of at least 10 contiguous nucleotides orbases of SEQ ID NO:185. It is also encompassed that not all the 8cytosines of SEQ ID NO:57 are modified as represented in SEQ ID NO:185.It is encompassed that 1, 2, 3, 4, 5, 6, or 7 of these cytosines aremodified.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

A preferred fragment of SEQ ID NO:57 comprises SEQ ID NO: 85 and apreferred fragment of SEQ ID NO:137 comprises SEQ ID NO: 165, each ofsaid fragments has a length of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32 or 33 nucleotides. Another preferred fragmentof SEQ ID NO:57 comprises SEQ ID NO: 86 and another preferred fragmentof SEQ ID NO:137 comprises SEQ ID NO: 166, each of said fragments has alength of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32 or 33 nucleotides.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO:186 and has a length of 24, 25,        26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment        of SEQ ID NO:186 comprising or consisting of at least 10        contiguous nucleotides or bases of SEQ ID NO:186. Such fragment        has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18,        19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides. It is also encompassed that not all the 7 uraciles        of SEQ ID NO:57 are modified as represented in SEQ ID NO:186. It        is encompassed that 1, 2, 3, 4, 5 or 6 of these uraciles are        modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its adenines have been replaced by 2,6-diaminopurines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 187 and has a length of 24, 25,        26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment        of SEQ ID NO:187 comprising or consisting of at least 10        contiguous nucleotides or bases of SEQ ID NO:187. Such fragment        has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18,        19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides. It is also encompassed that not all the 5 adenines        of SEQ ID NO:57 are modified as represented in SEQ ID NO:187. It        is encompassed that 1, 2, 3 or 4 of these adenines are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines and        all its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 188 and has a length of 24, 25,        26, 27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a fragment        of SEQ ID NO:188 comprising or consisting of at least 10        contiguous nucleotides or bases of SEQ ID NO:188. Accordingly,        said oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 188 and has a length of 24, 25,        26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment        of SEQ ID NO:188 comprising or consisting of at least 10        contiguous nucleotides or bases of SEQ ID NO:188. Such fragment        has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18,        19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides. It is also encompassed that not all the 8 cytosines        and not all the 7 uraciles of SEQ ID NO:57 are modified as        represented in SEQ ID NO:188. It is encompassed that 1, 2, 3, 4,        5, 6 or 7 of these cytosines and-or 1, 2, 3, 4, 5 or 6 of these        uraciles are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines and        all its adenines have been replaced by 2,6-diaminopurines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 189 and has a length of 24, 25,        26, 27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a fragment        of SEQ ID NO:189 comprising or consisting of at least 10        contiguous nucleotides or bases of SEQ ID NO:189. Accordingly,        said oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 189 and has a length of 24, 25,        26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment        of SEQ ID NO:189 comprising or consisting of at least 10        contiguous nucleotides or bases of SEQ ID NO:189. Such fragment        has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18,        19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides. It is also encompassed that not all the 8 cytosines        and not all the 5 adenines of SEQ ID NO:57 are modified as        represented in SEQ ID NO:189. It is encompassed that 1, 2, 3, 4,        5, 6 or 7 of these cytosines and-or 1, 2, 3 or 4 of these        adenines are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its adenines have been replaced by 2,6-diaminopurines and        all its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 190 and has a length of 24, 25,        26, 27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a fragment        of SEQ ID NO:190 comprising or consisting of at least 10        contiguous nucleotides or bases of SEQ ID NO:190. Such fragment        has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18,        19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides. It is also encompassed that not all the 5 adenines        and not all the 7 uraciles of SEQ ID NO:57 are modified as        represented in SEQ ID NO:190. It is encompassed that 1, 2, 3 or        4 of these adenines and-or 1, 2, 3, 4, 5 or 6 of these uraciles        are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its adenines have been replaced by 2,6-diaminopurines, all        its cytosines have been replaced by 5-methylcytosines and all        its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 191 and has a length of 24, 25,        26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment        of SEQ ID NO:191 comprising or consisting of at least 10        contiguous nucleotides or bases of SEQ ID NO:191. Such fragment        has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18,        19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides. It is also encompassed that not all the 8 cytosines        and not all the 7 uraciles and not all the 5 adenines of SEQ ID        NO:57 are modified as represented in SEQ ID NO:191. It is        encompassed that 1, 2, 3, 4, 5, 6 or 7 of these cytosines and-or        1, 2, 3, 4, 5 or 6 of these uraciles and-or 1, 2, 3 or 4 of        these adenines are modified.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 58 and has a length of 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides, or by a fragment of SEQ ID NO:58 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:58.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:58 isrepresented by SEQ ID NO:138.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 138 and has a length 25, 26, 27, 28, 29, 30, 31,32 or 33 nucleotides, or by a fragment of SEQ ID NO:138 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:138.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

Such preferred oligonucleotide is also defined as follows:

-   -   comprises a 2′-O-methyl phosphorothioate RNA monomer or consists        of 2′-O-methyl phosphorothioate RNA and    -   is represented by a nucleotide or a base sequence comprising or        consisting of SEQ ID NO: 58 or 138 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a nucleotide or        a base sequence comprising or consisting of a fragment of SEQ ID        NO: 58 or 138, said fragment comprising or consisting of at        least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 contiguous nucleotides        or bases of SEQ ID NO:58 or 138.

More preferably, such oligonucleotide comprises a 5-methylpyrimidine(i.e. a 5-methylcytosine, and/or a 5-methyluracil) and/or a2,6-diaminopurine base as earlier defined herein.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 58 and has a length of 25, 26,        27, 28, 29, 30, 31, 32, or 33 nucleotides or by a fragment of        SEQ ID NO:58 comprising or consisting of at least 10 contiguous        nucleotides or bases of SEQ ID NO:58. Such fragment has        preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33        nucleotides.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 59 and has a length of 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides or by a fragment of SEQ ID NO:59 comprising or consisting ofat least 10 contiguous nucleotides or bases of SEQ ID NO:59. Suchfragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Such preferred oligonucleotide is also defined as follows:

-   -   comprises a 2′-O-methyl phosphorothioate RNA monomer or consists        of 2′-O-methyl phosphorothioate RNA and    -   is represented by a nucleotide or a base sequence comprising or        consisting of SEQ ID NO: 59 and has a length of 24, 25, 26, 27,        28, 29, 30, 31, 32, or 33 nucleotides or by a nucleotide        sequence comprising or consisting of a fragment of SEQ ID NO:        59, said fragment comprising or consisting of at least 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32 or 33 contiguous nucleotides or bases of SEQ        ID NO:59.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:59 isrepresented by SEQ ID NO:139 and a preferred fragment of SEQ ID NO:139is represented by SEQ ID NO: 167 or 168 or 169 or 170.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA and is represented by a nucleotide or a basesequence comprising SEQ ID NO: 139 and has a length 24, 25, 26, 27, 28,29, 30, 31, 32, or 33 nucleotides, or by a fragment of SEQ ID NO:139comprising or consisting of at least 10 contiguous nucleotides or basesof SEQ ID NO:139.

More preferably, such oligonucleotide comprises a 5-methylpyrimidine(i.e. a 5-methylcytosine, and/or a 5-methyluracil) and/or a2,6-diaminopurine base as earlier defined herein. Even more preferably,said oligonucleotide has all its cytosines and/or all its uracil and/orall its adenines that have been substituted or modified as definedherein.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 59 and has a length of 24, 25,        26, 27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a fragment        of SEQ ID NO:59 comprising or consisting of at least 10        contiguous nucleotides or bases of SEQ ID NO:59.

Accordingly, said oligonucleotide is represented by a nucleotide or abase sequence comprising SEQ ID NO: 192 and has a length of 24, 25, 26,27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a fragment of SEQ IDNO:192 comprising or consisting of at least 10 contiguous nucleotides orbases of SEQ ID NO:192. It is also encompassed that not all the 5cytosines of SEQ ID NO:59 are modified as represented in SEQ ID NO:192.It is encompassed that 1, 2, 3 or 4 of these cytosines are modified.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

A preferred fragment of SEQ ID NO:59 comprises SEQ ID NO: 87 and apreferred fragment of SEQ ID NO:139 comprises SEQ ID NO:167, each ofsaid fragments has a length of 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32 or 33 nucleotides. Another preferred fragment of SEQ ID NO:59comprises SEQ ID NO: 88 and another preferred fragment of SEQ ID NO:139comprises SEQ ID NO:168, each of said fragments has a length of 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32 or 33 nucleotides. Another preferred fragment of SEQ ID NO:59comprises SEQ ID NO: 89 and another preferred fragment of SEQ ID NO:139comprises SEQ ID NO:169, each of said fragments has a length of 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32 or 33 nucleotides. Another preferred fragment of SEQ ID NO:59comprises SEQ ID NO: 90 and another preferred fragment of SEQ ID NO:139comprises SEQ ID NO:170, each of said fragments has a length of 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32 or 33 nucleotides.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO:193 and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a        fragment of SEQ ID NO:193 comprising or consisting of at least        10 contiguous nucleotides or bases of SEQ ID NO:193. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides. It is also encompassed that not all the 6        uraciles of SEQ ID NO:59 are modified as represented in SEQ ID        NO:193. It is encompassed that 1, 2, 3, 4 or 5 of these uraciles        are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its adenines have been replaced by 2,6-diaminopurines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 194 and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides or by a        fragment of SEQ ID NO:194 comprising or consisting of at least        10 contiguous nucleotides or bases of SEQ ID NO:194. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides. It is also encompassed that not all the 6        adenines of SEQ ID NO:59 are modified as represented in SEQ ID        NO:194. It is encompassed that 1, 2, 3, 4 or 5 of these adenines        are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines and        all its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 195 and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a        fragment of SEQ ID NO:195 comprising or consisting of at least        10 contiguous nucleotides or bases of SEQ ID NO:195.        Accordingly, said oligonucleotide is represented by a nucleotide        or a base sequence comprising SEQ ID NO: 195 and has a length of        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides,        or by a fragment of SEQ ID NO:195 comprising or consisting of at        least 10 contiguous nucleotides or bases of SEQ ID NO:195. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides. It is also encompassed that not all the 5        cytosines and not all the 6 uraciles of SEQ ID NO:59 are        modified as represented in SEQ ID NO:195. It is encompassed that        1, 2, 3 or 4 of these cytosines and-or 1, 2, 3, 4 or 5 of these        uraciles are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its cytosines have been replaced by 5-methylcytosines and        all its adenines have been replaced by 2,6-diaminopurines,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 1% and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a        fragment of SEQ ID NO:196 comprising or consisting of at least        10 contiguous nucleotides or bases of SEQ ID NO:1%. Accordingly,        said oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 1% and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a        fragment of SEQ ID NO:196 comprising or consisting of at least        10 contiguous nucleotides or bases of SEQ ID NO:196. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides. It is also encompassed that not all the 5        cytosines and not all the 6 adenines of SEQ ID NO:59 are        modified as represented in SEQ ID NO:196. It is encompassed that        1, 2, 3 or 4 of these cytosines and/or 1, 2, 3, 4 or 5 of these        adenines are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its adenines have been replaced by 2,6-diaminopurines and        all its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 197 and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a        fragment of SEQ ID NO:197 comprising or consisting of at least        10 contiguous nucleotides or bases of SEQ ID NO:197. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides. It is also encompassed that not all the 6        adenines and not all the 6 uraciles of SEQ ID NO:59 are modified        as represented in SEQ ID NO:197. It is encompassed that 1, 2, 3,        4 or 5 of these adenines and/or 1, 2, 3, 4 or 5 of these        uraciles are modified.

More preferably, an oligonucleotide:

-   -   consists of 2′-O-methyl phosphorothioate RNA,    -   all its adenines have been replaced by 2,6-diaminopurines, all        its cytosines have been replaced by 5-methylcytosines and all        its uraciles have been replaced by 5-methyluraciles,    -   such oligonucleotide is represented by a nucleotide or a base        sequence comprising SEQ ID NO: 198 and has a length of 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides, or by a        fragment of SEQ ID NO:198 comprising or consisting of at least        10 contiguous nucleotides or bases of SEQ ID NO:198. Such        fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32        or 33 nucleotides. It is also encompassed that not all the 5        cytosines and not all the 6 uraciles and not all the 6 adenines        of SEQ ID NO:59 are modified as represented in SEQ ID NO:198. It        is encompassed that 1, 2, 3 or 4 of these cytosines and/or 1, 2,        3, 4 or 5 of these uraciles and/or 1, 2, 3, 4 or 5 of these        adenines are modified.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 60 and has a length of 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides, or by a fragment of SEQ ID NO:60 comprising or consistingof at least 10 contiguous nucleotides or bases of SEQ ID NO:60.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:60 isrepresented by SEQ ID NO:140.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 140 and has a length 24, 25, 26, 27, 28, 29, 30,31, 32 or 33 nucleotides, or by a fragment of SEQ ID NO:140 comprisingor consisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:140.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 61 and has a length of 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides or by a fragment of SEQ ID NO:61 comprising or consisting ofat least 10 contiguous nucleotides or bases of SEQ ID NO:61.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:61 isrepresented by SEQ ID NO:141.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 141 and has a length 24, 25, 26, 27, 28, 29, 30,31, 32 or 33 nucleotides or by a fragment of SEQ ID NO:141 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:141.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

In a preferred embodiment, an oligonucleotide comprises a 2′-O-methylphosphorothioate RNA monomer or consists of 2′-O-methyl phosphorothioateRNA and more preferably comprises a 5-methylpyrimidine (i.e. a5-methylcytosine, and/or a 5-methyluracil) and/or a 2,6-diaminopurinebase, is represented by a nucleotide or a base sequence comprising SEQID NO: 62 and has a length of 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33nucleotides or by a fragment of SEQ ID NO:62 comprising or consisting ofat least 10 contiguous nucleotides or bases of SEQ ID NO:62.

Accordingly, a non-modified oligonucleotide derived from SEQ ID NO:62 isrepresented by SEQ ID NO:142.

Accordingly, in a preferred embodiment, an oligonucleotide comprises a2′-O-methyl phosphorothioate RNA monomer or consists of 2′-O-methylphosphorothioate RNA is represented by a nucleotide or a base sequencecomprising SEQ ID NO: 142 and has a length 24, 25, 26, 27, 28, 29, 30,31, 32 or 33 nucleotides or by a fragment of SEQ ID NO:142 comprising orconsisting of at least 10 contiguous nucleotides or bases of SEQ IDNO:142.

Such fragment has preferably a length of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides.

Accordingly, more preferably, said oligonucleotide comprises a5-methylpyrimidine (i.e. a 5-methylcytosine, and/or a 5-methyluracil)and/or a 2,6-diaminopurine base. Accordingly, even more preferably, saidoligonucleotide has all its cytosines and/or all its uracil and/or allits adenines that have been substituted or modified as defined herein.

Composition

In a second aspect, there is provided a composition comprising anoligonucleotide as described in the previous section entitled“Oligonucleotide”. This composition preferably comprises or consists ofan oligonucleotide as described above.

In a preferred embodiment, said composition is for use as a medicament.Said composition is therefore a pharmaceutical composition. Apharmaceutical composition usually comprises a pharmaceutically acceptedcarrier, diluent and/or excipient. In a preferred embodiment, acomposition of the current invention comprises a compound as definedherein and optionally further comprises a pharmaceutically acceptableformulation, filler, preservative, solubilizer, carrier, diluent,excipient, salt, adjuvant and/or solvent. Such pharmaceuticallyacceptable carrier, filler, preservative, solubilizer, diluent, salt,adjuvant, solvent and/or excipient may for instance be found inRemington: The Science and Practice of Pharmacy, 20th Edition.Baltimore, Md.: Lippincott Williams & Wilkins, 2000. The compound asdescribed in the invention may possess at least one ionizable group. Anionizable group may be a base or acid, and may be charged or neutral. Anionizable group may be present as ion pair with an appropriatecounterion that carries opposite charge(s). Examples of cationiccounterions are sodium, potassium, cesium, Tris, lithium, calcium,magnesium, trialkylammonium, triethylammonium, and tetraalkylammonium.Examples of anionic counterions are chloride, bromide, iodide, lactate,mesylate, acetate, trifluoroacetate, dichloroacetate, and citrate.Examples of counterions have been described (e.g. Kumar. 2008, which isincorporated here in its entirety by reference).

In a preferred embodiment, a composition comprises the oligonucleotideof the invention and sodium as counterion. Said oligonucleotide presentin said composition may also be named as an oligonucleotide in itssodium form.

In another preferred embodiment, a composition comprises theoligonucleotide of the invention and calcium and/or magnesium ascounterion. Said oligonucleotide present in said composition may also benamed as an oligonucleotide in its calcium or magnesium or mixedcalcium/magnesium form.

Such type of composition comprising an oligonucleotide of the inventionand a counterion may be obtained through either formulating thecounterion salt of the oligonucleotide or by adding appropriate amountsof said salt to an oligonucleotide. A positive effect of calcium saltspresent in the composition comprising an oligonucleotide with respect toimmunostimulatory effects of said oligonucleotides has been described(e.g. patent application WO 2012021985 (Replicor), incorporated here inits entirety by reference).

A pharmaceutical composition may comprise an aid in enhancing thestability, solubility, absorption, bioavailability, activity,pharmacokinetics, pharmacodynamics and cellular uptake of said compound,in particular an excipient capable of forming complexes, nanoparticles,microparticles, nanotubes, nanogels, hydrogels, poloxamers or pluronics,polymersomes, colloids, microbubbles, vesicles, micelles, lipoplexes,and/or liposomes. Examples of nanoparticles include polymericnanoparticles, gold nanoparticles, magnetic nanoparticles, silicananoparticles, lipid nanoparticles, sugar particles, proteinnanoparticles and peptide nanoparticles.

A preferred composition comprises at least one excipient that mayfurther aid in enhancing the targeting and/or delivery of saidcomposition and/or said oligonucleotide to a tissue and/or a cell and/orinto a tissue and/or a cell. A preferred tissue or cell is a muscletissue or cell.

Many of these excipients are known in the art (e.g. see Bruno, 2011) andmay be categorized as a first type of excipient. Examples of first typeof excipients include polymers (e.g. polyethyleneimine (PEI),poly-2-hydroxypropyleneimine (pHP), polypropyleneimine (PPI), dextranderivatives, butylcyanoacrylate (PBCA), hexylcyanoacrylate (PHCA),poly(lactic-co-glycolic acid) (PLGA), polyamines (e.g. spermine,spermidine, putrescine, cadaverine), chitosan, poly(amido amines)(PAMAM), poly(ester amine), polyvinyl ether, polyvinyl pyrrolidone(PVP), polyethylene glycol (PEG) cyclodextrins, hyaluronic acid,colominic acid, and derivatives thereof), dendrimers (e.g.poly(amidoamine)), lipids (e.g. 1,2-dioleoyl-3-dimethylammonium propane(DODAP), dioleoyldimethylammonium chloride (DODAC), phosphatidylcholinederivatives [e.g. 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)],lyso-phosphatidylcholine derivaties [e.g.1-stearoyl-2-lyso-sn-glycero-3-phosphocholine (S-LysoPC)],sphingomyeline,2-{3-[Bis-(3-amino-propyl)-amino]-propylamino}-N-ditetracedyl carbamoylmethylacetamide (RPR209120), phosphoglycerol derivatives [e.g.1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, sodium salt (DPPG-Na),phosphaticid acid derivatives [1,2-distearoyl-sn-glycero-3-phosphaticidacid, sodium salt (DSPA), phosphatidylethanolamine derivatives [e.g.dioleoyl-L-R-phosphatidylethanolamine (DOPE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine(DSPE),2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE),],N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP),N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA),1,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propylamid (DOSPER),(1,2-dimyristyolxypropyl-3-dimethylhydroxy ethyl ammonium (DMRIE),(N1-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN),dimethyldioctadecylammonium bromide (DDAB),1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC),(b-L-Arginyl-2,3-L-diaminopropionic acid-N-palmityl-N-olelyl-amidetrihydrochloride (AtuFECTO1), 1, N,N-dimethyl-3-aminopropane derivatives[e.g. 1,2-distearoyloxy-N,N-dimethyl-3-aminopropane (DSDMA),1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DoDMA),1,2-Dilinoleyloxy-N,N-3-dimethylaminopropane (DLinDMA),2,2-dilinoleyl-4-dimethylaminomethyl [1,3]-dioxolane (DLin-K-DMA),phosphatidylserine derivatives[1,2-dioleyl-sn-glycero-3-phospho-L-serine, sodium salt (DOPS)],cholesterol} proteins (e.g. albumin, gelatins, atellocollagen), andpeptides (e.g. protamine, PepFects, NickFects, polyarginine, polylysine,CADY, MPG).

Another preferred composition may comprise at least one excipientcategorized as a second type of excipient. A second type of excipientmay comprise or contain a conjugate group as described herein to enhancetargeting and/or delivery of the composition and/or of theoligonucleotide of the invention to a tissue and/or cell and/or into atissue and/or cell, as for example muscle tissue or cell. Both types ofexcipients may be combined together into one single composition asidentified herein.

The skilled person may select, combine and/or adapt one or more of theabove or other alternative excipients and delivery systems to formulateand deliver a compound for use in the present invention.

Such a pharmaceutical composition of the invention may be administeredin an effective concentration at set times to an animal, preferably amammal. More preferred mammal is a human being. An oligonucleotide or acomposition as defined herein for use according to the invention may besuitable for direct administration to a cell, tissue and/or an organ invivo of individuals affected by or at risk of developing a disease orcondition as identified herein, and may be administered directly invivo, ex vivo or in vitro. Administration may be via topical, systemicand/or parenteral routes, for example intravenous, subcutaneous,intraperitoneal, intrathecal, intramuscular, ocular, nasal, urogenital,intradermal, dermal, enteral, intravitreal, intracavemous,intracerebral, intrathecal, epidural or oral route.

Preferably, such a pharmaceutical composition of the invention may beencapsulated in the form of an emulsion, suspension, pill, tablet,capsule or soft-gel for oral delivery, or in the form of aerosol or drypowder for delivery to the respiratory tract and lungs.

In an embodiment an oligonucleotide of the invention may be usedtogether with another compound already known to be used for thetreatment of said disease. Such other compounds may be used for reducinginflammation, preferably for reducing muscle tissue inflammation, and/oran adjunct compound for improving muscle fiber function, integrityand/or survival and/or improve, increase or restore cardiac function.Examples are, but not limited to, a steroid, preferably a(gluco)corticosteroid, an ACE inhibitor (preferably perindopril), anangiotensin II type 1 receptor blocker (preferably losartan), a tumornecrosis factor-alpha (TNFα) inhibitor, a TGFβ inhibitor (preferablydecorin), human recombinant biglycan, a source of mIGF-1, a myostatininhibitor, mannose-6-phosphate, an antioxidant, an ion channelinhibitor, a protease inhibitor, a phosphodiesterase inhibitor(preferably a PDE5 inhibitor, such as sildenafil or tadalafil), ahistone deacetylase inhibitor (HDAC inhibitor, androgen receptormodulator, creatine, creatine phosphate, and/or L-arginine. Suchcombined use may be a sequential use: each component is administered ina distinct composition. Alternatively, each compound may be usedtogether in a single composition.

Use

In a further aspect, there is provided the use of a composition or anoligonucleotide as described in the previous sections for use as amedicament or part of therapy, or applications in which saidoligonucleotide exerts its activity intracellularly.

Preferably, an oligonucleotide or composition of the invention is foruse as a medicament or part of a therapy for preventing, delaying,curing, ameliorating and/or treating DMD or BMD.

Method

In a further aspect, there is provided a method for preventing,treating, curing, ameliorating and/or delaying a condition or disease asdefined in the previous section in an individual, in a cell, tissue ororgan of said individual. The method comprising administering anoligonucleotide or a composition of the invention to said individual ora subject in the need thereof.

The method according to the invention wherein an oligonucleotide or acomposition as defined herein may be suitable for administration to acell, tissue and/or an organ in vivo of individuals affected by any ofthe herein defined diseases, and may be administered in vivo, ex vivo orin vitro. An individual or a subject in need is preferably a mammal,more preferably a human being.

In a further aspect, there is provided a method for diagnosis whereinthe oligonucleotide of the invention is provided with a radioactivelabel or fluorescent label.

In an embodiment, in a method of the invention, a concentration of anoligonucleotide or composition is ranged from 0.01 nM to 1 μM. Morepreferably, the concentration used is from 0.05 to 500 nM, or from 0.1to 500 nM, or from 0.02 to 500 nM, or from 0.05 to 500 nM, even morepreferably from 1 to 200 nM.

Dose ranges of an oligonucleotide or composition according to theinvention are preferably designed on the basis of rising dose studies inclinical trials (in vivo use) for which rigorous protocol requirementsexist. An oligonucleotide as defined herein may be used at a dose whichis ranged from 0.01 to 200 mg/kg or 0.05 to 100 mg/kg or 0.1 to 50 mg/kgor 0.1 to 20 mg/kg, preferably from 0.5 to 10 mg/kg.

The ranges of concentration or dose of oligonucleotide or composition asgiven above are preferred concentrations or doses for in vitro or exvivo uses. The skilled person will understand that depending on theidentity of the oligonucleotide used, the target cell to be treated, thegene target and its expression levels, the medium used and thetransfection and incubation conditions, the concentration or dose ofoligonucleotide used may further vary and may need to be optimised anyfurther.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, the verb “to consist” may be replaced by“to consist essentially of” meaning that an oligonucleotide or acomposition as defined herein may comprise additional component(s) thanthe ones specifically identified, said additional component(s) notaltering the unique characteristic of the invention. In addition,reference to an element by the indefinite article “a” or “an” does notexclude the possibility that more than one of the element is present,unless the context clearly requires that there be one and only one ofthe elements. The indefinite article “a” or “an” thus usually means “atleast one”.

Each embodiment as identified herein may be combined together unlessotherwise indicated. All patent and literature references cited in thepresent specification are hereby incorporated by reference in theirentirety.

Definitions

Throughout the application, the word “binds”, “targets”, “hybridizes”could be used interchangeably when used in the context of an antisenseoligonucleotide which is reverse complementary to a part of a pre-mRNAas identified herein.

In addition, throughout the application, the expression “able to bind”,“able to target”, “able to hybridize” could be used interchangeably whenused in the context of an antisense oligonucleotide which is reversecomplementary to a part of a pre-mRNA as identified herein and for whichconditions could be found wherein said oligonucleotide could bind,target or hybridize with said part of said pre-mRNA.

As used herein, “hybridization” refers to the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases). For example,the natural base adenine is nucleobase complementary to the naturalnucleobases thymine and uracil which pair through the formation ofhydrogen bonds. The natural base guanine is nucleobase complementary tothe natural bases cytosine and 5-methylcytosine. Hybridization can occurunder varying circumstances.

As used herein, “specifically hybridizes” refers to the ability of anoligomeric compound to hybridize to one nucleic acid site with greateraffinity than it hybridizes to another nucleic acid site. In certainembodiments, an anti sense oligonucleotide specifically hybridizes tomore than one target site.

In the context of the invention, “hybridizes” is used underphysiological conditions in a cell, preferably a muscular cell unlessotherwise indicated.

As used herein, “nucleoside” refers to a compound comprising aheterocyclic base moiety and a sugar moiety. Nucleosides include, butare not limited to, naturally occurring nucleosides (as found in DNA andRNA), abasic nucleosides, modified nucleosides, and sugar-modifiednucleosides. Nucleosides may be modified with any of a variety ofsubstituents.

As used herein, “sugar moiety” means a natural (furanosyl), a modifiedsugar moiety or a sugar surrogate.

As used herein, “modified sugar moiety” means a chemically-modifiedfuranosyl sugar or a non-furanosyl sugar moiety. Also, embraced by thisterm are furanosyl sugar analogs and derivatives including tricyclicsugars, bicyclic sugars, tetrahydropyrans, morpholinos, 2′-modifiedsugars, 4′-modified sugars, 5′-modified sugars, and 4′-substitutedsugars.

As used herein, “sugar-modified nucleoside” means a nucleosidecomprising a modified sugar moiety.

As used herein the term “sugar surrogate” refers to a structure that iscapable of replacing the furanose ring of a naturally occurringnucleoside. In certain embodiments, sugar surrogates are non-furanose(or 4′-substituted furanose) rings or ring systems or open systems. Suchstructures include simple changes relative to the natural furanose ring,such as a six membered ring or may be more complicated as is the casewith the non-ring system used in peptide nucleic acid. Sugar surrogatesincludes without limitation morpholinos and cyclohexenyls andcyclohexitols. In most nucleosides having a sugar surrogate group theheterocyclic base moiety is generally maintained to permithybridization.

As used herein, “nucleotide” refers to a nucleoside further comprising amodified or unmodified phosphate linking group or a non-phosphateinternucleoside linkage.

As used herein, “linked nucleosides” may or may not be linked byphosphate linkages and thus includes “linked nucleotides”.

As used herein, “nucleobase” refers to the heterocyclic base portion ofa nucleoside.

Nucleobases may be naturally occurring or may be modified and thereforeinclude, but are not limited to adenine, cytosine, guanine, uracil,thymine and analogues thereof such as 5-methylcytosine. In certainembodiments, a nucleobase may comprise any atom or group of atomscapable of hydrogen bonding to a base of another nucleic acid.

As used herein, “modified nucleoside” refers to a nucleoside comprisingat least one modification compared to naturally occurring RNA or DNAnucleosides. Such modification may be at the sugar moiety and/or at thenucleobases.

As used herein, “Tm” means melting temperature which is the temperatureat which the two strands of a duplex nucleic acid separate. Tm is oftenused as a measure of duplex stability or the binding affinity of anantisense compound toward a complementary RNA molecule.

As used herein, “2′-modified” or “2′-substituted” refers to a nucleosidecomprising a sugar comprising a substituent at the 2′ position otherthan H or OH. 2′-modified nucleosides include, but are not limited to,bicyclic nucleosides wherein the bridge connecting two carbon atoms ofthe sugar ring connects the 2′ carbon and another carbon of the sugarring; and nucleosides with non-bridging 2′-substituents, such as allyl,amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), orO—CH₂—C(═O)—N(R_(m))(R_(n)), wherein each R_(m) and R_(n) is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.2′-modified nucleosides may further comprise other modifications, forexample at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-OMe” or “2′-OCH₃” or “2′-O-methyl” each refers to anucleoside comprising a sugar comprising an —OCH₃ group at the 2′position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′-OCH₂CH₂OCH₃” or“2′-O-methoxyethyl” each refers to a nucleoside comprising a sugarcomprising a —OCH₂CH₂OCH₃ group at the 2′ position of the sugar ring.

As used herein, the term “adenine analogue” means a chemically-modifiedpurine nucleobase that, when incorporated into an oligomer, is capablewith forming a Watson-Crick base pair with either a thymine or uracil ofa complementary strand of RNA or DNA.

As used herein, the term “uracil analogue” means a chemically-modifiedpyrimidine nucleobase that, when incorporated into an oligomer, iscapable with forming a Watson-Crick base pair with either a adenine of acomplementary strand of RNA or DNA.

As used herein, the term “thymine analogue” means a chemically-modifiedpyrimidine nucleobase that, when incorporated into an oligomer, iscapable with forming a Watson-Crick base pair with an adenine of acomplementary strand of RNA or DNA.

As used herein, the term “cytosine analogue” means a chemically-modifiedpyrimidine nucleobase that, when incorporated into an oligomer, iscapable with forming a Watson-Crick base pair with a guanine of acomplementary strand of RNA or DNA. For example, cytosine analogue canbe a 5-methylcytosine.

As used herein, the term “guanine analogue” means a chemically-modifiedpurine nucleobase that, when incorporated into an oligomer, is capablewith forming a Watson-Crick base pair with a cytosine of a complementarystrand of RNA or DNA.

As used herein, the term “guanosine” refers to a nucleoside orsugar-modified nucleoside comprising a guanine or guanine analognucleobase.

As used herein, the term “uridine” refers to a nucleoside orsugar-modified nucleoside comprising a uracil or uracil analognucleobase.

As used herein, the term “thymidine” refers to a nucleoside orsugar-modified nucleoside comprising a thymine or thymine analognucleobase.

As used herein, the term “cytidine” refers to a nucleoside orsugar-modified nucleoside comprising a cytosine or cytosine analognucleobase.

As used herein, the term “adenosine” refers to a nucleoside orsugar-modified nucleoside comprising an adenine or adenine analognucleobase.

As used herein, “oligonucleotide” refers to a compound comprising aplurality of linked nucleosides. In certain embodiments, one or more ofthe plurality of nucleosides is modified. In certain embodiments, anoligonucleotide comprises one or more ribonucleosides (RNA) and/ordeoxyribonucleosides (DNA).

As used herein “oligonucleoside” refers to an oligonucleotide in whichnone of the internucleoside linkages contains a phosphorus atom. As usedherein, oligonucleotides include oligonucleosides.

As used herein, “modified oligonucleotide” or “chemically-modifiedoligonucleotide” refers to an oligonucleotide comprising at least onemodified sugar, a modified nucleobase and/or a modified internucleosidelinkage or backbone.

As used herein, “internucleoside linkage” or “backbone” refers to acovalent linkage between adjacent nucleosides.

As used herein “naturally occurring internucleoside linkage” refers to a3′ to 5′ phosphodiester linkage.

As used herein, “modified internucleoside linkage” refers to anyinternucleoside linkage other than a naturally occurring internucleosidelinkage.

As used herein, “oligomeric compound” refers to a polymeric structurecomprising two or more sub-structures. In certain embodiments, anoligomeric compound is an oligonucleotide. In certain embodiments, anoligomeric compound is a single-stranded oligonucleotide. In certainembodiments, an oligomeric compound is a double-stranded duplexcomprising two oligonucleotides. In certain embodiments, an oligomericcompound is a single-stranded or double-stranded oligonucleotidecomprising one or more conjugate groups and/or terminal groups.

As used herein, “conjugate” refers to an atom or group of atoms bound toan oligonucleotide or oligomeric compound. In general, conjugate groupsmodify one or more properties of the compound to which they areattached, including, but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and clearance. Conjugate groups are routinely used in thechemical arts and are linked directly or via an optional linking moietyor linking group to the parent compound such as an oligomeric compound.In certain embodiments, conjugate groups includes without limitation,intercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, thioethers, polyethers, cholesterols, thiocholesterols, cholicacid moieties, folate, lipids, phospholipids, biotin, phenazine,phenanthridine, anthraquinone, adamantane, acridine, fluoresceins,rhodamines, coumarins and dyes. In certain embodiments, conjugates areterminal groups. In certain embodiments, conjugates are attached to a 3′or 5′ terminal nucleoside or to an internal nucleosides of anoligonucleotide.

As used herein, “conjugate linking group” refers to any atom or group ofatoms used to attach a conjugate to an oligonucleotide or oligomericcompound. Linking groups or bifunctional linking moieties such as thoseknown in the art are amenable to the present invention.

As used herein, “antisense compound” refers to an oligomeric compound,at least a portion of which is at least partially complementary to atarget nucleic acid to which it hybridizes and modulates the activity,processing or expression of said target nucleic acid.

As used herein, “expression” refers to the process by which a geneultimately results in a protein. Expression includes, but is not limitedto, transcription, splicing, post-transcriptional modification, andtranslation.

As used herein, “antisense oligonucleotide” refers to an antisensecompound that is an oligonucleotide.

As used herein, “antisense activity” refers to any detectable and/ormeasurable activity attributable to the hybridization of an anti sensecompound to its target nucleic acid.

In certain embodiments, such activity may be an increase or decrease inan amount of a nucleic acid or protein. In certain embodiments, suchactivity may be a change in the ratio of splice variants of a nucleicacid or protein. Detection and/or measuring of antisense activity may bedirect or indirect. In certain embodiments, antisense activity isassessed by observing a phenotypic change in a cell or animal.

As used herein, “target nucleic acid” refers to any nucleic acidmolecule the expression, amount, or activity of which is capable ofbeing modulated by an antisense compound. In certain embodiments, thetarget nucleic acid is DNA or RNA. In certain embodiments, the targetRNA is mRNA, pre-mRNA, non-coding RNA, pri-microRNA, pre-microRNA,mature microRNA, promoter-directed RNA, or natural antisensetranscripts. For example, the target nucleic acid can be a cellular gene(or mRNA transcribed from the gene) whose expression is associated witha particular disorder or disease state, or a nucleic acid molecule froman infectious agent. In certain embodiments, target nucleic acid is aviral or bacterial nucleic acid.

As used herein, “target mRNA” refers to a pre-selected RNA molecule thatencodes a protein.

As used herein, “targeting” or “targeted to” refers to the associationof an antisense compound to a particular target nucleic acid molecule ora particular region of nucleotides within a target nucleic acidmolecule. An anti sense compound targets a target nucleic acid if it issufficiently complementary to the target nucleic acid to allowhybridization under physiological conditions.

As used herein, “target site” refers to a region of a target nucleicacid that is bound by an antisense compound. In certain embodiments, atarget site is at least partially within the 3′ untranslated region ofan RNA molecule. In certain embodiments, a target site is at leastpartially within the 5′ untranslated region of an RNA molecule. Incertain embodiments, a target site is at least partially within thecoding region of an RNA molecule. In certain embodiments, a target siteis at least partially within an exon of an RNA molecule. In certainembodiments, a target site is at least partially within an intron of anRNA molecule. In certain embodiments, a target site is at leastpartially within a microRNA target site of an RNA molecule. In certainembodiments, a target site is at least partially within a repeat regionof an RNA molecule.

As used herein, “target protein” refers to a protein, the expression ofwhich is modulated by an antisense compound. In certain embodiments, atarget protein is encoded by a target nucleic acid. In certainembodiments, expression of a target protein is otherwise influenced by atarget nucleic acid.

As used herein, “complementarity” in reference to nucleobases refers toa nucleobase that is capable of base pairing with another nucleobase.For example, in DNA, adenine (A) is complementary to thymine (T). Forexample, in RNA, adenine (A) is complementary to uracil (U). In certainembodiments, complementary nucleobase refers to a nucleobase of anantisense compound that is capable of base pairing with a nucleobase ofits target nucleic acid. For example, if a nucleobase at a certainposition of an antisense compound is capable of hydrogen bonding with anucleobase at a certain position of a target nucleic acid, then theposition of hydrogen bonding between the oligonucleotide and the targetnucleic acid is considered to be complementary at that nucleobase pair.Nucleobases comprising certain modifications may maintain the ability topair with a counterpart nucleobase and thus, are still capable ofnucleobase complementarity.

As used herein, “non-complementary” in reference to nucleobases refersto a pair of nucleobases that do not form hydrogen bonds with oneanother or otherwise support hybridization.

As used herein, “complementary” in reference to linked nucleosides,oligonucleotides, or nucleic acids, refers to the capacity of anoligomeric compound to hybridize to another oligomeric compound ornucleic acid through nucleobase complementarity. In certain embodiments,an antisense compound and its target are complementary to each otherwhen a sufficient number of corresponding positions in each molecule areoccupied by nucleobases that can bond with each other to allow stableassociation between the anti sense compound and the target. One skilledin the art recognizes that the inclusion of mismatches is possiblewithout eliminating the ability of the oligomeric compounds to remain inassociation. Therefore, described herein are antisense compounds thatmay comprise up to about 20% nucleotides that are mismatched (i.e., arenot nucleobase complementary to the corresponding nucleotides of thetarget). Preferably the antisense compounds contain no more than about15%, more preferably not more than about 10%, most preferably not morethan 5% or no mismatches. The remaining nucleotides are nucleobasecomplementary or otherwise do not disrupt hybridization (e.g., universalbases). One of ordinary skill in the art would recognize the compoundsprovided herein are at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%complementary to a target nucleic acid.

As used herein, “modulation” refers to a perturbation of amount orquality of a function or activity when compared to the function oractivity prior to modulation. For example, modulation includes thechange, either an increase (stimulation or induction) or a decrease(inhibition or reduction) in gene expression. As a further example,modulation of expression can include perturbing splice site selection ofpre-mRNA processing, resulting in a change in the amount of a particularsplice-variant present compared to conditions that were not perturbed.As a further example, modulation includes perturbing translation of aprotein.

As used herein, “motif” refers to a pattern of modifications in anoligomeric compound or a region thereof. Motifs may be defined bymodifications at certain nucleosides and/or at certain linking groups ofan oligomeric compound.

As used herein, “nucleoside motif” refers to a pattern of nucleosidemodifications in an oligomeric compound or a region thereof. Thelinkages of such an oligomeric compound may be modified or unmodified.Unless otherwise indicated, motifs herein describing only nucleosidesare intended to be nucleoside motifs. Thus, in such instances, thelinkages are not limited.

As used herein, “linkage motif” refers to a pattern of linkagemodifications in an oligomeric compound or region thereof. Thenucleosides of such an oligomeric compound may be modified orunmodified. Unless otherwise indicated, motifs herein describing onlylinkages are intended to be linkage motifs. Thus, in such instances, thenucleosides are not limited.

As used herein, “the same modifications” refer to modifications relativeto naturally occurring molecules that are the same as one another,including absence of modifications. Thus, for example, two unmodifiedDNA nucleoside have “the same modification,” even though the DNAnucleoside is unmodified.

As used herein, “type of modification” in reference to a nucleoside or anucleoside of a “type” refers to the modification of a nucleoside andincludes modified and unmodified nucleosides. Accordingly, unlessotherwise indicated, a “nucleoside having a modification of a firsttype” may be an unmodified nucleoside.

As used herein, “separate regions” refers to a portion of an oligomericcompound wherein the nucleosides and internucleoside linkages within theregion all comprise the same modifications; and the nucleosides and/orthe internucleoside linkages of any neighboring portions include atleast one different modification.

As used herein, “pharmaceutically acceptable salts” refers to salts ofactive compounds that retain the desired biological activity of theactive compound and do not impart undesired toxicological effectsthereto.

As used herein, “cap structure” or “terminal cap moiety” refers tochemical modifications incorporated at either terminus of an anti sensecompound.

As used herein, the term “independently” means that each occurrence of arepetitive variable within a claimed oligonucleotide is selectedindependent of one another. For example, each repetitive variable can beselected so that (i) each of the repetitive variables are the same, (ii)two or more are the same, or (iii) each of the repetitive variables canbe different.

General Chemistry Definitions

As used herein, “alkyl” refers to a saturated straight or branchedhydrocarbon substituent or radical, typically containing up totwenty-four carbon atoms. Examples of alkyl groups include, but are notlimited to, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl,decyl, dodecyl and the like. Alkyl groups typically include from 1 to 24carbon atoms, more typically from 1 to 12 carbon atoms (C₁-C₁₂ alkyl)with from 1 to 6 carbon atoms (C₁-C₆ alkyl) being more preferred. Theterm “lower alkyl” as used herein includes from 1 to 6 carbon atoms(C₁-C₆ alkyl). Alkyl groups as used herein may optionally include one ormore further substituent groups.

As used herein, “alkenyl” refers to a straight or branched hydrocarbonchain radical or substituent, typically containing up to twenty-fourcarbon atoms, and having at least one carbon-carbon double bond.Examples of alkenyl groups include, but are not limited to, ethenyl,propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadienyland the like. Alkenyl groups typically include from 2 to 24 carbonatoms, more typically from 2 to 12 carbon atoms with from 2 to 6 carbonatoms being more preferred. Alkenyl groups as used herein may optionallyinclude one or more further substituent groups.

As used herein, “alkynyl” refers to a straight or branched hydrocarbonradical or substituent, typically containing up to twenty-four carbonatoms, and having at least one carbon-carbon triple bond. Examples ofalkynyl groups include, but are not limited to, ethynyl, 1-propynyl,1-butynyl, and the like. Alkynyl groups typically include from 2 to 24carbon atoms, more typically from 2 to 12 carbon atoms with from 2 to 6carbon atoms being more preferred. Alkynyl groups as used herein mayoptionally include one or more further substituent groups.

As used herein, “aminoalkyl” refers to an amino substituted alkylradical or substituent. This term is meant to include C₁-C₁₂ alkylgroups having an amino substituent at any position and wherein theaminoalkyl group is attached to the parent molecule via its alkylmoiety. The alkyl and/or amino portions of the aminoalkyl group can befurther substituted with substituent groups.

As used herein, “aliphatic” refers to a straight or branched hydrocarbonradical or substituent, typically containing up to twenty-four carbonatoms, wherein the saturation between any two carbon atoms is a single,double or triple bond. An aliphatic group preferably contains from 1 to24 carbon atoms, more typically from 1 to 12 carbon atoms with from 1 to6 carbon atoms being more preferred. The straight or branched chain ofan aliphatic group may be interrupted with one or more heteroatoms thatinclude nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groupsinterrupted by heteroatoms include without limitation polyalkoxys, suchas polyalkylene glycols, polyamines, and polyimines. Aliphatic groups asused herein may optionally include further substituent groups.

As used herein, “alicyclic” or “alicyclyl” refers to a cyclic radical orsubstituent, wherein the ring system is aliphatic. The ring system cancomprise one or more rings wherein at least one ring is aliphatic.Preferred alicyclic moieties include rings having from 5 to 9 carbonatoms in the ring. Alicyclic groups as used herein may optionallyinclude further substituent groups.

As used herein, “alkoxy” refers to a radical or substituent comprisingan alkyl group and an oxygen atom, wherein the alkoxy group is attachedto a parent molecule via its oxygen atom. Examples of alkoxy groupsinclude, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy,n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy andthe like. Alkoxy groups as used herein may optionally include furthersubstituent groups.

As used herein, “halo”, “halide” and “halogen” refer to an atom, radicalor substituent selected from fluorine, chlorine, bromine and iodine.

As used herein, “aryl” and “aromatic” refer to a radical or substituentcomprising a mono- or polycyclic carbocyclic ring system having one ormore aromatic rings. Examples of aryl groups include, but are notlimited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl andthe like. Preferred aryl ring systems have from 5 to 20 carbon atoms inone or more rings. Aryl groups as used herein may optionally includefurther substituent groups.

As used herein, “aralkyl” and “arylalkyl” refer to a radical orsubstituent comprising an alkyl group and an aryl group, wherein thearalkyl or arylalkyl group is attached to a parent molecule via itsalkyl moiety. Examples include, but are not limited to, benzyl,phenethyl and the like. Aralkyl groups as used herein may optionallyinclude further substituent groups attached to the alkyl, the aryl orboth groups that form the radical or substituent.

As used herein, “heterocyclyl” refers to a radical or substituentcomprising a mono- or polycyclic ring system that includes at least oneheteroatom and is unsaturated, partially saturated or fully saturated,thereby including heteroaryl groups. Heterocyclyl is also meant toinclude fused ring system moieties wherein one or more of the fusedrings contain at least one heteroatom and the other rings can containone or more heteroatoms or optionally contain no heteroatoms. Aheterocyclic group typically includes at least one atom selected fromsulfur, nitrogen or oxygen. Examples of heterocyclic groups include[1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl,morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl,pyridazinonyl, tetrahydrofuryl and the like. Heterocyclic groups as usedherein may optionally include further substituent groups.

As used herein, “heteroaryl” and “heteroaromatic” refer to a radical orsubstituent comprising a mono- or polycyclic aromatic ring, ring systemor fused ring system wherein at least one of the rings is aromatic andincludes one or more heteroatom. Heteroaryl is also meant to includefused ring systems including systems where one or more of the fusedrings contain no heteroatoms. Heteroaryl groups typically include onering atom selected from sulfur, nitrogen or oxygen. Examples ofheteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl,pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and thelike. Heteroaryl radicals or substituents can be attached to a parentmolecule directly or through a linking moiety such as an aliphatic groupor a heteroatom. Heteroaryl groups as used herein may optionally includefurther substituent groups.

As used herein, “heteroarylalkyl” refers to a radical or substituentcomprising a heteroaryl group as previously defined and an alkyl moiety,wherein the heteroarylalkyl group is attached to a parent molecule viaits alkyl moiety. Examples include, but are not limited to,pyridinylmethyl, pyrimidinylethyl, napthyridinylpropyl and the like.Heteroarylalkyl groups as used herein may optionally include furthersubstituent groups on one or both of the heteroaryl or alkyl portions.

As used herein, “mono or polycyclic” refers to any ring systems, such asa single ring or a polycyclic system having rings that are fused orlinked, and is meant to be inclusive of single and mixed ring systemsindividually selected from aliphatic, alicyclic, aryl, heteroaryl,aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic andheteroarylalkyl. Such mono and polycyclic structures can contain ringsthat have a uniform or varying degree of saturation, including fullysaturated, partially saturated or fully unsaturated rings. Each ring cancomprise ring atoms selected from C, N, O and S to give rise toheterocyclic rings as well as rings comprising only C ring atoms.Heterocyclic and all-carbon rings can be present in a mixed motif, suchas for example benzimidazole wherein one ring of the fused ring systemhas only carbon ring atoms and the other ring has two nitrogen atoms.The mono or polycyclic structures can be further substituted withsubstituent groups such as for example phthalimide which has two oxogroups (═O) attached to one of the rings. In another aspect, mono orpolycyclic structures can be attached to a parent molecule directlythrough a ring atom, through a substituent group or a bifunctionallinking moiety.

As used herein, “acyl” refers to a radical or substituent comprising acarbonyl moiety (C═O or —C(O)—) and a further substituent X, wherein theacyl group is attached to a parent molecule via its carbonyl moiety. Assuch, an acyl group is formally obtained by removal of a hydroxyl groupfrom an organic acid and has the general formula —C(O)—X, wherein X istypically aliphatic, alicyclic or aromatic. The term “acyl” is alsomeant to include heteroacyl radicals or substituents with generalformula —Y(O)_(n)—X, wherein X is as defined above and Y(O)_(n) istypically sulfonyl, sulfinyl or phosphate. Examples of acyl groupsinclude aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls,aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallyinclude further substituent groups.

As used herein, “substituent” and “substituent group” include groupsthat are typically added to other substituents or parent compounds toenhance desired properties or give desired effects. Substituent groupscan be protected or unprotected and can be attached to one availablesite or to many available sites in a parent compound. Substituent groupsmay also be further substituted with other substituent groups and may beattached directly or via a linking group such as an alkyl or hydrocarbylgroup to a parent compound. Herein, “hydrocarbyl” refers to any groupcomprising C, O and H. Included are straight, branched and cyclic groupshaving any degree of saturation. Such hydrocarbyl groups can include oneor more heteroatoms selected from N, O and S and can be furthersubstituted with one or more substituent groups.

Unless otherwise indicated, the term substituted or “optionallysubstituted” refers to the optional presence of any of the followingsubstituents: halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl(—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)), aliphatic groups, alicyclicgroups, alkoxy, substituted oxo (—O—R_(aa)), aryl, aralkyl,heterocyclic, heteroaryl, heteroarylalkyl, amino (—NR_(bb)R_(cc)), imino(═NR_(bb)), amido (—C(O)NR_(bb)R_(cc) or —N(R_(bb))C(O)R_(aa)), azido(—N₃), nitro (—NO₂), cyano (—CN), carbamido (—OC(O)NR_(bb)R_(cc) or—N(R_(bb))C(O)OR_(aa)), ureido (—N(R_(bb))C(O)NR_(bb)R_(cc)), thioureido(—N(R_(bb))C(S)NR_(bb)R_(cc)), guanidinyl(—N(R_(bb))C(═NR_(bb))NR_(bb)R_(cc)), amidinyl(—C(═NR_(bb))NR_(bb)R_(cc) or —N(R_(bb))C(NR_(bb))R_(aa)), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)),sulfonamidyl (—S(O)₂NR_(bb)R_(cc) or —N(R_(bb))S(O)₂R_(bb)) andconjugate groups. Herein, each R_(aa), R_(bb) and R_(cc) is,independently, H, an optionally linked chemical functional group or afurther substituent group, preferably but without limitation chosen fromthe group consisting of H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy,acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic andheteroarylalkyl. Selected substituents within the compounds describedherein are present to a recursive degree.

In this context, “recursive substituent” means that a substituent mayrecite another instance of itself. Because of the recursive nature ofsuch substituents, theoretically, a large number may be present in anygiven claim. One of ordinary skill in the art of medicinal chemistry andorganic chemistry understands that the total number of such substituentsis reasonably limited by the desired properties of the compoundintended. Such properties include, by way of example and not limitation,physical properties such as molecular weight, solubility or log P,application properties such as activity against the intended target andpractical properties such as ease of synthesis.

Recursive substituents are an intended aspect of the invention. One ofordinary skill in the art of medicinal and organic chemistry understandsthe versatility of such substituents. To the degree that recursivesubstituents are present in a claim of the invention, the total numberwill be determined as set forth above.

The terms “stable compound” and “stable structure” as used herein aremeant to indicate a compound that is sufficiently robust to surviveisolation to a useful degree of purity from a reaction mixture, andformulation into an efficacious therapeutic agent. Only stable compoundsare contemplated herein.

As used herein, a zero (0) in a range indicating number of a particularunit means that the unit may be absent. For example, an oligomericcompound comprising 0-2 regions of a particular motif means that theoligomeric compound may comprise one or two such regions having theparticular motif, or the oligomeric compound may not have any regionshaving the particular motif. In instances where an internal portion of amolecule is absent, the portions flanking the absent portion are bounddirectly to one another. Likewise, the term “none” as used herein,indicates that a certain feature is not present.

As used herein, “analogue” or “derivative” means either a compound ormoiety similar in structure but different in respect to elementalcomposition from the parent compound regardless of how the compound ismade. For example, an analogue or derivative compound does not need tobe made from the parent compound as a chemical starting material.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show a comparison of AONs with or without cytosine to5-methylcytosine substitution in differentiated healthy muscle cells invitro after transfection with (FIG. 1A) PS229L/PS524, SEQ ID NO:52(corresponding to SEQ ID NO: 91 for the non-modified sequence,corresponding to SEQ ID NO: 92 wherein all cytosines are modified) or(FIG. 1B) PS220/PS339 (SEQ ID NO:21, corresponding to SEQ ID NO:101 forthe non-modified sequence, corresponding to SEQ ID NO:200 wherein allcytosines are modified) or (FIG. 1C) PS524/PS1317/PS1318/PS1319, SEQ IDNO:52 (corresponding to SEQ ID NO: 92 (PS524) wherein all 6 cytosinesare modified, to SEQ ID NO: 217 (PS1317) wherein 4 of the 6 cytosinesare modified, to SEQ ID NO: 218 (PS1318) wherein 2 of the 6 cytosinesare modified and to SEQ ID NO:219 (PS1319) wherein 3 of the 6 cytosinesare modified SEQ ID NO:217). Average skipping percentages werecalculated from triplo (n=3) (FIG. 1A-B) or duplo (n=2) (FIG. 1C)transfections per concentration. Solid lines refer to AONs with5-methylcytosines, dotted lines to AONs with non-substituted cytosines(FIGS. 1A-B).

FIGS. 2A-B are a summary of the pharmacokinetic study in wild type(control) and mdx mice, comparing plasma and muscle tissue profiles ofAONs with 5-methylcytosines (PS524, SEQ ID NO:52 (i.e. corresponding toSEQ ID NO: 92 wherein all cytosines are modified) and PS652, SEQ IDNO:57 (i.e. corresponding to SEQ ID NO: 185 wherein all cytosines aremodified) and AONs with unmodified (non-methylated) cytosines (PS229L,SEQ ID NO:52 corresponding to SEQ ID NO: 91 for the non-modifiedsequence, and PS531, SEQ ID NO:57 corresponding to SEQ ID NO: 137 forthe non-modified sequence). (FIG. 2A) Pharmacokinetic tissue analysisof: 1) the ratio between the average levels of AON in muscle in mdx miceversus control mice after one single sc injection; 2) the levels of theAONs (sg/g) in several mdx muscles (dia=diaphragm,gastroc=gastrocnemius, quadr=quadriceps, tric=triceps) at 14 days; 3)the relative muscle/kidney and muscle/liver levels at day 14, and 4) theestimated half-life of the different AONs in triceps. (FIG. 2B)Pharmacokinetic plasma analysis of 1) Tmax (time at which Cmax wasreached, only two time points of analysis included (15 or 60 min), 2)Cmax (highest plasma concentration reached), 3) AUC (area under curve;indicative for bioavailability) and 4) Cl (plasma clearance at 24 h.

FIGS. 3A-H show an analysis of cytokine levels in human whole blood uponincubation with 0, 10, 25, or 50 μg/ml of AONs with unmodified cytosinesPS232 (SEQ ID NO: 39, corresponding to SEQ ID NO: 119 for thenon-modified sequence) and PS534 (SEQ ID NO:59, corresponding to SEQ IDNO: 139 for the non-modified sequence) (black bars) or AONs with5-methylcytosines PS648 (SEQ ID NO: 39, corresponding to SEQ ID NO: 201wherein all cytosines are modified) and PS653 (SEQ ID NO:59, to SEQ IDNO: 192 wherein all cytosines are modified) (grey bars). The levels ofTNFα (FIGS. 3A,B), MCP-1 (FIGS. 3C,D), IP-10 (FIGS. 3E,F), and IL6(FIGS. 3G,H) were determined using commercially available ELISA kits.Each experiment was repeated four times (n=4). Data is shown for themost pronounced response of each cytokine.

FIGS. 4A-B show activity comparisons of AONs with 5-methylcytosinesand/or 5-methyluracils with corresponding AONs without these basemodifications, (FIG. 4A) Transfection of 200 nM, in duplo, intodifferentiated healthy muscle cells in vitro. Activity was expressed asaverage percentage exon 51 (PS43, non-modified sequence represented bySEQ ID NO: 111, PS559 corresponding to SEQ ID NO: 202, wherein alluraciles are modified, PS1106 corresponding to SEQ ID NO:203, whereinall cytosines and all uraciles are modified. All sequences are derivedfrom SEQ ID NO: 31), exon 44 (PS188, non-modified sequence representedby SEQ ID NO: 95, PS785, corresponding to SEQ ID NO: 204, wherein alluraciles are modified, PS1107: corresponding to SEQ ID NO:205, whereinall cytosines and all uraciles are modified. All sequences are derivedfrom SEQ ID NO 15); or exon 52 (PS235, non-modified sequence representedby SEQ ID NO: 120, PS786: corresponding to SEQ ID NO: 172, wherein alluraciles are modified. All sequences are derived from SEQ ID NO 40)skipping (n=2). AON sequences (5′ to 3′) and base modifications (bold,underlined nucleotides) are shown in the table underneath. (FIG. 4B)Intramuscular injection of 20 μg of PS49 (non-modified sequence, SEQ IDNO: 216) or PS959 (modified sequence wherein all uracils are modified,SEQ ID NO:214) in the gastrocnemius muscles of mdx mice. Activity wasexpressed as average percentage murine exon 23 skipping (n=4). AONsequences (5′ to 3′) and base modifications (bold, underlinednucleotides) are shown in the table underneath.

FIGS. 5A-C show activity comparisons of AONs with 2,6-diaminopurineswith corresponding AONs without this base modification. (FIG. 5A),Transfection of 200 nM, in duplo, into differentiated healthy musclecells in vitro. Activity was expressed as average percentage exon 51(PS43, non-modified sequence represented by SEQ ID NO: 111, PS403,corresponding to SEQ ID NO: 206, wherein all adenines have beenmodified. All sequences are derived from SEQ ID NO: 31), exon 52 (PS235,non-modified sequence represented by SEQ ID NO: 120, PS897:corresponding to SEQ ID NO: 173, wherein all adenines have beenmodified. All sequences are derived from SEQ ID NO: 40), or exon 44(PS188, non-modified sequence represented by SEQ ID NO: 95, PS733:corresponding to SEQ ID NO: 207, wherein all adenines have beenmodified. All sequences are derived from SEQ ID NO: 15) skipping (n=2).AON sequences (5′ to 3′) and base modifications (bold, underlinednucleotides) are shown in the table underneath. (FIG. 5B) and (FIG. 5C)The effect of substituting all unmodified adenines (PS188; SEQ ID NO:95) with 2,6-diaminopurines (PS733; SEQ ID NO:207) on in vitro safety.As markers for activation of the alternative complement pathway, splitfactors C3a (FIG. 5B) and Bb (FIG. 5C) were measured in monkey plasma.

EXAMPLES

TABLE 1General structures of AONs. X = C or m⁵C, Y = U or m⁵U, Z = A or a²A;I = inosine (hypoxanthine base), X₁ = m⁵C, Y₁ = m⁵U, Z₁ = a²A DMD ExonAON Sequence (5′→ 3′) SEQ ID NO 44 GXXZYYYXYXZZXZGZYXY  14GCCAUUUCUCAACAGAUCU  94 44 YXZGXYYXYGYYZGXXZXYG  15 UCAGCUUCUGUUAGCCACUG 95 Y₁CAGCY₁Y₁CY₁GY₁Y₁AGCCACY₁G 204 UX₁AGX₁UUX₁UGUUAGX₁X₁AX₁UG 208Y₁X₁AGX₁Y₁Y₁X₁Y₁GY₁Y₁AGX₁X₁AX₁Y₁G 205 UCZ₁GCUUCUGUUZ₁GCCZ₁CUG 207 44YYYGYZYYYZGXZYGYYXXX  16 UUUGUAUUUAGCAUGUUCCC  96 44ZYYXYXZGGZZYYYGYGYXYYYX  17 AUUCUCAGGAAUUUGUGUCUUUC  97 44XXZYYYGYZYYYZGXZYGYYXXX  18 CCAUUUGUAUUUAGCAUGUUCCC  98 44YXYXZGGZZYYYGYGYXYYYX  19 UCUCAGGAAUUUGUGUCUUUC  99 44GXXZYYYXYXZZXZGZYXYGYXZ  20 GCCAUUUCUCAACAGAUCUGUCA 100 45YYYGXXGXYGXXXZZYGXXZYXXYG  21 UUUGCCGCUGCCCAAUGCCAUCCUG 101UUUGX₁X₁GX₁UGX₁X₁X₁AAUGX₁X₁AUX₁X₁UG 200Y₁Y₁Y₁GX₁X₁GX₁Y₁GX₁X₁X₁AAY₁GX₁X₁AY₁X₁X₁Y₁G 209UUUGCCGCUGCCCZ₁Z₁UGCCZ₁UCCUG 210 45 YYGXXGXYGXXXZZYGXXZYXXYG  22UUGCCGCUGCCCAAUGCCAUCCUG 102 45 YYGXXGXYGXXXZZYGXXZYXXYGG  23UUGCCGCUGCCCAAUGCCAUCCUGG 103 45 YGXXGXYGXXXZZYGXXZYXXYG  24UGCCGCUGCCCAAUGCCAUCCUG 104 45 YGXXGXYGXXXZZYGXXZYXXYGG  25UGCCGCUGCCCAAUGCCAUCCUGG 105 45 GXXGXYGXXXZZYGXXZYXXYG  26GCCGCUGCCCAAUGCCAUCCUG 106 45 XXGXYGXXXZZYGXXZYXXYGG  27CCGCUGCCCAAUGCCAUCCUGG 107 45 YYYGXXIXYGXXXZZYGXXZYXXYG  28UUUGCCICUGCCCAAUGCCAUCCUG 108 45 XZGYYYGXXGXYGXXXZZYGXXZYX  29CAGUUUGCCGCUGCCCAAUGCCAUC 109 45 XZGYYYGXXGXYGXXXZZYGXXZYXXYGGZ  30CAGUUUGCCGCUGCCCAAUGCCAUCCUGGA 110 51 YXZZGGZZGZYGGXZYYYXY  31UCAAGGAAGAUGGCAUUUCU 111 Y₁CAAGGAAGAY₁GGCAY₁Y₁Y₁CY₁ 202Y₁X₁AAGGAAGAY₁GGX₁AY₁Y₁Y₁X₁Y₁ 203 UCZ₁Z₁GGZ₁Z₁GZ₁UGGCZ₁UUUCU 206UX₁AAGGAAGAUGGX₁AUUUX₁U 215 51 YGGXZYYYXYZGYYYGG  32 UGGCAUUUCUAGUUUGG112 51 XZYXZZGGZZGZYGGXZYYYXY  33 CAUCAAGGAAGAUGGCAUUUCU 113 51XZZXZYXZZGGZZGZYGGXZYYYXY  34 CAACAUCAAGGAAGAUGGCAUUUCU 114 51XXYXYGYGZYYYYZYZZXYYGZY  35 CCUCUGUGAUUUUAUAACUUGAU 115 51XXZGZGXZGGYZXXYXXZZXZYX  36 CCAGAGCAGGUACCUCCAACAUC 116 51ZXZYXZZGGZZGZYGGXZYYYXYZGYYYGG  37 ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG 117 51ZXZYXZZGGZZGZYGGXZYYYXYZG  38 ACAUCAAGGAAGAUGGCAUUUCUAG 118 52XYXYYGZYYGXYGGYXYYGYYYYYX  39 CUCUUGAUUGCUGGUCUUGUUUUUC 119X₁UX₁UUGAUUGX₁UGGUX₁UUGUUUUUX₁ 201 52 GGYZZYGZGYYXYYXXZZXYGG  40GGUAAUGAGUUCUUCCAACUGG 120 GGUAAUGAGUUX₁UUX₁X₁AAX₁UGG 171GGY₁AAY₁GAGY₁Y₁CY₁Y₁CCAACY₁GG 172 GGUZ₁Z₁UGZ₁GUUCUUCCZ₁Z₁CUGG 173GGY₁AAY₁GAGY₁Y₁X₁Y₁Y₁X₁X₁AAX₁Y₁GG 174 GGUZ₁Z₁UGZ₁GUUX₁UUX₁X₁Z₁Z₁X₁UGG175 GGY₁Z₁Z₁Y₁GZ₁GY₁Y₁CY₁Y₁CCZ₁Z₁CY₁GG 176GGY₁Z₁Z₁Y₁GZ₁GY₁Y₁X₁Y₁Y₁X₁X₁Z₁Z₁X₁Y₁GG 177 52 YXYYGZYYGXYGGYXYYGYYYYYXZ 41 UCUUGAUUGCUGGUCUUGUUUUUCA 121 52 YYXXZZXYGGGGZXGXXYXYGYYXX  42UUCCAACUGGGGACGCCUCUGUUCC 122 52 YGYYXYZGXXYXYYGZYYGXYGGYX  43UGUUCUAGCCUCUUGAUUGCUGGUC 123 UGUUX₁UAGX₁X₁UX₁UUGAUUGX₁UGGUX₁ 178Y₁GY₁Y₁CY₁AGCCY₁CY₁Y₁GAY₁Y₁GCY₁GGY₁C 179 UGUUCUZ₁GCCUCUUGZ₁UUGCUGGUC 180Y₁GY₁Y₁X₁Y₁AGX₁X₁Y₁X₁Y₁Y₁GAY₁Y₁GX₁Y₁GGY₁X₁ 181UGUUX₁UZ₁GX₁X₁UX₁UUGZ₁UUGX₁UGGUX₁ 182Y₁GY₁Y₁CY₁Z₁GCCY₁CY₁Y₁GZ₁Y₁Y₁GCY₁GGY₁C 183Y₁GY₁Y₁X₁Y₁Z₁GX₁X₁Y₁X₁Y₁Y₁GZ₁Y₁Y₁GX₁Y₁GGY₁X₁ 184 53 XYGYYGXXYXXGGYYXYG 44 CUGUUGCCUCCGGUUCUG 124 53 XZZXYGYYGXXYXXGGYYXYGZ  45CAACUGUUGCCUCCGGUUCUGA 125 53 XZZXYGYYGXXYXXGGYYXYGZZ  46CAACUGUUGCCUCCGGUUCUGAA 126 53 XZZXYGYYGXXYXXGGYYXYGZZG  47CAACUGUUGCCUCCGGUUCUGAAG 127 53 XYGYYGXXYXXGGYYXYGZZGG  48CUGUUGCCUCCGGUUCUGAAGG 128 53 XYGYYGXXYXXGGYYXYGZZGGY  49CUGUUGCCUCCGGUUCUGAAGGU 129 53 XYGYYGXXYXXGGYYXYGZZGGYG  50CUGUUGCCUCCGGUUCUGAAGGUG 130 53 XYGYYGXXYXXGGYYXYGZZGGYGY  51CUGUUGCCUCCGGUUCUGAAGGUGU 131 53 GYYGXXYXXGGYYXYGZZGGYGYYX  52GUUGCCUCCGGUUCUGAAGGUGUUC  91 GUUGX₁X₁UX₁X₁GGUUX₁UGAAGGUGUUX₁  92UUGX₁X₁UCCGGUUX₁UGAAGGUGUUX₁ 217 GUUGX₁X₁UCCGGUUCUGAAGGUGUUC 218GUUGCX₁UCCGGUUX₁UGAAGGUGUUX₁ 219 GY₁Y₁GCCY₁CCGGY₁Y₁CY₁GAAGGY₁GY₁Y₁C 211GY₁Y₁GX₁X₁Y₁X₁X₁GGY₁Y₁X₁Y₁GAAGGY₁GY₁Y₁X₁ 212 GUUGCCUCCGGUUCUGZ₁Z₁GGUGUUC213 53 GXXYXXGGYYXYGZZGGYGYYXYYG  53 GCCUCCGGUUCUGAAGGUGUUCUUG 133 53YYGXXYXXGGYYXYGZZGGYGYYXYYGYZX  54 UUGCCUCCGGUUCUGAAGGUGUUCUUGUAC 134 53XYGYYGXXYXXGGYYXYGZZGGYGYYXYYG  55 CUGUUGCCUCCGGUUCUGAAGGUGUUCUUG 135 53XZZXYGYYGXXYXXGGYYXYGZZGGYGYYXYYG  56 CAACUGUUGCCUCCGGUUCUGAAGGUGUUCUUG136 55 GZGYYYXYYXXZZZGXZGXXYXYX  57 GAGUUUCUUCCAAAGCAGCCUCUC 137GAGUUUX₁UUX₁X₁AAAGX₁AGX₁X₁UX₁UX₁ 185 GAGY₁Y₁Y₁CY₁Y₁CCAAAGCAGCCY₁CY₁C 186GZ₁GUUUCUUCCZ₁Z₁Z₁GCZ₁GCCUCUC 187 GAGY₁Y₁Y₁X₁Y₁Y₁X₁X₁AAAGX₁AGX₁X₁YA₁YA₁188 GZ₁GUUUX₁UUX₁X₁Z₁Z₁Z₁GX₁Z₁GX₁XXXXX₁ 189GZ₁GY₁Y₁Y₁CY₁Y₁CCZ₁Z₁Z₁GCZ₁GCCY₁CY₁C 190GZ₁GY₁Y₁Y₁X₁Y₁Y₁X₁X₁Z₁Z₁Z₁GX₁Z₁GX₁X₁Y₁X₁Y₁X₁ 191 55YZYGZGYYYXYYXXZZZGXZGXXYX  58 UAUGAGUUUCUUCCAAAGCAGCCUC 138 55ZGXZYXXYGYZGGZXZYYGGXZGY  59 AGCAUCCUGUAGGACAUUGGCAGU 139AGX₁AUX₁X₁UGUAGGAX₁AUUGGX₁AGU 192 AGCAY₁CCY₁GY₁AGGACAY₁Y₁GGCAGY₁ 193Z₁GCZ₁UCCUGUZ₁GGZ₁CZ₁UUGGCZ₁GU 194 AGX₁AY₁X₁X₁Y₁GY₁AGGAX₁AY₁Y₁GGX₁AGY₁195 Z₁GX₁Z₁UX₁X₁UGUZ₁GGZ₁X₁Z₁UUGGX₁Z₁GU 196Z₁GCZ₁Y₁CCY₁GY₁Z₁GGZ₁CZ₁Y₁Y₁GGCZ₁GY₁ 197Z₁GX₁Z₁Y₁X₁X₁Y₁GY₁Z₁GGZ₁X₁Z₁Y₁Y₁GGX₁Z₁GY₁ 198 55XZYXXYGYZGGZXZYYGGXZGYYG  60 CAUCCUGUAGGACAUUGGCAGUUG 140 55YXXYGYZGGZXZYYGGXZGYYGYY  61 UCCUGUAGGACAUUGGCAGUUGUU 141 55XYGYZGGZXZYYGGXZGYYGYYYX  62 CUGUAGGACAUUGGCAGUUGUUUC 142

TABLE 2General structures of AONs. X = C or m⁵C, Y = U or m⁵U, Z = A or a²A;I = inosine (hypoxanthine base), X₁ = m⁵C, Y₁ = m⁵U, Z₁ = a²A DMD ExonAON Sequence (5′→3′) SEQ ID NO 44 ZYYYXYXZZXZGZ  63 AUUUCUCAACAGA 143 44ZGXYYXYGYYZGXXZ  64 AGCUUCUGUUAGCCA 144 44 ZYYXYXZGGZZ  65 AUUCUCAGGAA145 44 ZYYYGYZYYYZGXZ  66 AUUUGUAUUUAGCA 146 44 ZYYYXYXZZXZGZYXYGYXZ  67AUUUCUCAACAGAUCUGUCA 147 44 ZYYYXYXZZXZGZ  68 AUUUCUCAACAGA 148 44ZXZGZYXYGYXZ  69 ACAGAUCUGUCA 149 45 YYYGXXGXYGXXXZZYGXXZ  70UUUGCCGCUGCCCAAUGCCA 150 45 XGXYGXXXZZYGXXZYXXYG  71CGCUGCCCAAUGCCAUCCUG 151 45 GXXGXYGXXXZZYGXXZYXX  72GCCGCUGCCCAAUGCCAUCC 152 51 ZZGGZZGZYGGXZ  73 AAGGAAGAUGGCA 153 51ZGGZZGZYGGXZ  74 AGGAAGAUGGCA 154 51 ZGZGXZGGYZ  75 AGAGCAGGUA 155 51ZGXZGGYZXXYXXZ  76 AGCAGGUACCUCCA 156 51 ZXXYXXZZXZ  77 ACCUCCAACA 15752 ZZYGZGYYXYYXXZZ  78 AAUGAGUUCUUCCAA 158 52 ZYGZGYYXYYXXZ  79AUGAGUUCUUCCA 159 52 ZGYYXYYXXZ  80 AGUUCUUCCA 160 52 ZGXXYXYYGZ  81AGCCUCUUGA 161 53 GYYGXXYXXGGYYXYGZZGG  82 GUUGCCUCCGGUUCUGAAGG 162 53XYXXGGYYXYGZZGGYGYYX  83 CUCCGGUUCUGAAGGUGUUC 163 53 XXYXXGGYYXYGZZGGY 84 CCUCCGGUUCUGAAGGU 164 55 ZGYYYXYYXXZZZGXZ  85 AGUUUCUUCCAAAGCA 16555 ZGYYYXYYXXZ  86 AGUUUCUUCCA 166 55 ZGXZYXXYGYZGGZXZYYGGXZ  87AGCAUCCUGUAGGACAUUGGCA 167 55 ZGXZYXXYGYZ  88 AGCAUCCUGUA 168 55ZYXXYGYZGGZ  89 AUCCUGUAGGA 169 55 ZGGZXZYYGGXZ  90 AGGACAUUGGCA 170

TABLE 3 MOST PREFERRED AONSGeneral structures of AONs. X = C or m⁵C, Y = U or m⁵U, Z = A or a²A;I = inosine (hypoxanthine base), X₁ = m⁵C, Y₁ = m⁵U, Z₁ = a²A DMD ExonAON Sequence (5′→3′) SEQ ID NO 44 YXZGXYYXYGYYZGXXZXYG  15UCAGCUUCUGUUAGCCACUG  95 PS188 FIG. 4,5 Y₁CAGCY₁Y₁CY₁GY₁Y₁AGCCACY₁G 204PS785 FIG. 4 UX₁AGX₁UUX₁UGUUAGX₁X₁AX₁UG 208 PS658Y₁X₁AGX₁Y₁Y₁X₁Y₁GY₁Y₁AGX₁X₁AX₁Y₁G 205 PS1107 FIG. 4UCZ₁GCUUCUGUUZ₁GCCZ₁CUG 207 PS733 FIG. 5 45 YYYGXXGXYGXXXZZYGXXZYXXYG 21 UUUGCCGCUGCCCAAUGCCAUCCUG 101 PS220 FIG. 1bUUUGX₁X₁GX₁UGX₁X₁X₁AAUGX₁X₁AUX₁X₁UG 200 PS399 FIG. 1bY₁Y₁Y₁GX₁X₁GX₁Y₁GX₁X₁X₁AAY₁GX₁X₁AY₁X₁X₁Y₁G 209 PS1108UUUGCCGCUGCCCZ₁Z₁UGCCZUCCUG 210 PS1229 YYYGXXIXYGXXXZZYGXXZYXXYG  28UUUGCCICUGCCCAAUGCCAUCCUG 108 PS305 51 YXZZGGZZGZYGGXZYYYXY  31UCAAGGAAGAUGGCAUUUCU 111 PS43 FIG. 4, 5 Y₁CAAGGAAGAY₁GGCAY₁Y₁Y₁CY₁ 202PS559 FIG. 4 Y₁X₁AAGGAAGAY₁GGX₁AY₁Y₁Y₁X₁Y₁ 203 PS1106 FIG. 4UCZ₁Z₁GGZ₁Z₁GZ₁UGGCZ₁UUUCU 206 PS403 FIG. 5 UX₁AAGGAAGAUGGX₁AUUUX₁U 215PS401 52 GGYZZYGZGYYXYYXXZZXYGG  40 GGUAAUGAGUUCUUCCAACUGG 120 PS235FIG. 4, 5 GGUAAUGAGUUX₁UUX₁X₁AAX₁UGG 171 PS650GGYAAY₁GAGY₁Y₁CY₁Y₁CCAACY₁GG 172 PS786 FIG. 4GGUZ₁Z₁UGZ₁GUUCUUCCZ₁Z₁CUGG 173 PS897 FIG. 5GGYAAY₁GAGY₁Y₁X₁Y₁Y₁X₁X₁AAX₁Y₁GG 174 PS1110 53 GYYGXXYXXGGYYXYGZZGGYGYYX 52 GUUGCCUCCGGUUCUGAAGGUGUUC  91 PS229L FIG. 1a, 2GUUGX₁X₁UX₁X₁GGUUX₁UGAAGGUGUUX₁  92 PS524 FIG. 1a, c, 2GUUGX₁X₁UCCGGUUX₁UGAAGGUGUUX₁ 217 PS1317 FIG. 1cGUUGX₁X₁UCCGGUUCUGAAGGUGUUC 218 PS1318 FIG. 1cGUUGCX₁UCCGGUUX₁UGAAGGUGUUX₁ 219 PS1319 FIG. 1cGY₁Y₁GCCY₁CCGGY₁Y₁CY₁GAAGGY₁GY₁Y₁C 211GY₁Y₁GX₁X₁Y₁X₁X₁GGY₁Y₁X₁Y₁GAAGGY₁GY₁Y₁X₁ 212 PS1109GUUGCCUCCGGUUCUGZ₁Z₁GGUGUUC 213 55 GZGYYYXYYXXZZZGXZGXXYXYX  57GAGUUUCUUCCAAAGCAGCCUCUC 137 PS531 FIG. 2GAGUUUX₁UUX₁X₁AAAGX₁AGX₁X₁UX₁UX₁ 185 PS652 FIG. 2GAGY₁Y₁Y₁CY₁Y₁CCAAAGCAGCCY₁CY₁C 186 GZ₁GUUUCUUCCZ₁Z₁Z₁GCZ₁GCCUCUC 187GAGY₁Y₁Y₁X₁Y₁Y₁X₁X₁AAAGX₁AGX₁X₁Y₁X₁Y₁X₁ 188 PS1112

Preferred non modified oligonucleotides (X=C, Y=U, Z=A) are morepreferably derived from each of the oligonucleotide basis sequence (SEQID NO: 14-90) and are represented by a nucleotide or base sequence SEQID NO:91, 93-170.

Preferred modified oligonucleotides derived from one of the nucleotideor base sequences SEQ ID NO: 14-90 and comprising at least one X is m⁵Cand/or at least one Y is m⁵U and/or at least one Z is a²A arerepresented by a nucleotide or abase sequence comprising or consistingof SEQ ID NO: 92, 171-213, 215, 217, 218, 219. Even more preferredmodified oligonucleotides (all X=m⁵C=X₁ and/or all Y=m⁵U=Y₁ and/or allZ=a²A=Z₁) are derived from the most preferred nucleotide or basesequences (SEQ ID NO: 15, 21, 31, 40, 52, and 57) and are represented bySEQ ID NO: 92, 171-174, 185-188, 199, 200, 202-213, 215, 217, 218, 219.The most preferred modified oligonucleotides are disclosed in Table 3.

Example 1 Material and Methods AONs

All oligonucleotides (PS220/PS399, based on SEQ ID NO:21 correspondingto SEQ ID NO:101 for the non-modified sequence (PS220) and to SEQ IDNO:200 wherein all cytosines are modified (PS399);PS229L/PS524/PS1317/PS1318/PS1319, based on SEQ ID NO:52 correspondingto SEQ ID NO:91 for the non-modified sequence (PS229L), to SEQ ID NO:92(PS524) wherein all 6 cytosines are modified, to SEQ ID NO: 217 (PS1317)wherein 4 of the 6 cytosines are modified, to SEQ ID NO: 218 (PS1318)wherein 2 of the 6 cytosines are modified and to SEQ ID NO:219 (PS1319)wherein 3 of the 6 cytosines are modified; PS232/PS648, based on SEQ IDNO: 39 corresponding to SEQ ID NO:119 for the non-modified sequence(PS232) and to SEQ ID NO:201 wherein all cytosines are modified (PS648);PS531/PS652, based on SEQ ID NO:57 corresponding to SEQ ID NO:137 forthe non-modified sequence (PS531) and to SEQ ID NO:185 wherein allcytosines are modified (PS652); PS534/PS653, based on SEQ ID NO:59corresponding to SEQ ID NO:139 for the non-modified sequence (PS534) andto SEQ ID NO:192 wherein all cytosines are modified (PS653)) were2′-O-methyl phosphorothioate RNA, and synthesized using an OP-10synthesizer (GE/ÄKTA Oligopilot), through standard phosphoramiditeprotocols, or obtained from commercial suppliers, in 40 nmol-4.5 mmolsynthesis scale. Prosensa-synthesized oligonucleotides were cleaved anddeprotected in a two step sequence (DIEA followed by conc. NH₄OHtreatment), purified by HPLC and dissolved in water and an excess ofNaCl was added to exchange ions. After evaporation, compounds wereredissolved in water, desalted by FPLC or ultrafiltration andlyophilized. Mass spectrometry confirmed the identity of all compounds,and purity (determined by UPLC) was found acceptable for all compounds(>75-80%); compounds obtained from commercial sources were used asreceived: PS399 (ChemGenes, 1 μmol synthesis scale, used as received),PS1317, PS1318, and PS1319 (ChemGenes, 200 nmol synthesis scale, used asreceived), PS229L, PS232, PS524, and PS648 (EuroGentec, 40 nmolsynthesis scale, used as received), PS229L (Prosensa, 5.9 g obtainedmaterial, purity 81%), PS524 (Avecia, 4.5 mmol synthesis scale, purity93%), PS534 (Prosensa, 2 μmol synthesis scale, purity 86%), PS653(Prosensa, 40 nmol synthesis scale, purity 77%), PS531 (Avecia, 4.6 gobtained material, purity 85%), PS652 (Avecia, 2.4 g obtained material,purity 84% and 3.8 g obtained material, purity 82%). For the in vitrotransfection experiments described herein, 50 μM working solutions ofthe AONs were prepared in 20 mM phosphate buffer (pH 7.0). For the wholeblood cytokine release assays in this example, the concentrations of thestock solutions (prepared in DNase/RNase-free distilled water(Invitrogen)) varied: PS232 (8.75 mg/mL), PS534 (7.02 mg/mL), PS648(8.55 mg/mL), PS653 (8.12 mg/mL).

Transfection and RT-PCR Analysis

Differentiated human healthy control muscle cells (myotubes) weretransfected in 6-wells plates with a triplo AON concentration series of0-100-200-400 nM (FIG. 1A, PS229L/PS524, SEQ ID NO:91/92) or0-50-100-200-400-800 nM (FIG. 1B, PS220/PS399, SEQ ID NO: 101/200) orwith an in duplo concentration of 400 nM (FIG. 1C,PS524/PS1317/PS1318/PS1319, SEQ ID NO:92/217/218/219), according tonon-GLP standard operating procedures. For transfection polyethylenimine(ExGen500, Fermentas) was used (2 μl per μg AON, in 0.15M NaCl).Aforementioned transfection procedures were adapted from previouslyreported material and methods (Aartsma-Rus et al., 2003). At 24 hrsafter transfection, RNA was isolated and analyzed by RT-PCR. Briefly, togenerate dystrophin-specific cDNA, a DMD gene specific reverse primer inexon 47 (PS220/PS399) or exon 55 (PS229L/PS524/PS1317/PS1318/PS1319) wasused in the reverse transcriptase (RT) reaction on 1000 ng input RNA.The PCR analysis was subsequently done on 3 l of dystrophin cDNA foreach sample, and included a first and nested PCR using DMD gene specificprimers in exons flanking exon 45 (PS220/PS399) or 53(PS229L/PS524/PS1317/PS1318/PS1319). The RNA isolation and RT-PCRanalysis were performed according to non-GLP standard operatingprocedures as described (Aartsma-Rus et al., 2003). RT-PCR products wereanalyzed by gel electrophoresis (2% agarose gels). The resulting RT-PCRfragments were quantified through DNA Lab-on-a-Chip analysis (Agilent).The data was processed by “Agilent 2100 Bioanalyzer” software and Excel2007. The ratio of the smaller transcript product (containing the exon45 (PS220/PS399) or 53 skip (PS229L/PS524/PS1317/PS1318/PS1319)) to thetotal amount of transcript products was assessed (representing the exon45 or 53 skipping efficiencies in percentages) and directly compared tothat in non-transfected cells.

Pharmacokinetic Study in Wild Type and Mdx Mice

Mdx (C57Bl/10ScSn-Dmd^(mdx)/J) and wild-type (C57Bl/10ScSnJ) mice at 5weeks of age were obtained from Jackson Laboratory (Maine USA). The AONs(PS229L/PS524 corresponding to SEQ ID NO: 91/92, PS531/PS652corresponding to SEQ ID NO: 137/185) were administered in physiologicalsaline at a dose of 100 mg/kg by subcutaneous injections three times perweek for two weeks. To determine the plasma profile of the AONs, plasmasamples were taken from 2 animals per time-point (per AON group) at thefollowing times for the animals: 15 min, 1 h, 2 h, 6 h and 24 hoursafter dosing. To obtain plasma, venous whole blood was collected intoLi-Heparin tubes, centrifuged and kept at −80° C. until analysis. Fordistribution analysis 7 organs (heart, kidney cortex, liver, diaphragm,gastrocnemius, quadriceps & triceps) were harvested upon sacrifice ofthe animals. The tissues were snap frozen and stored at −80° C. untilanalysis.

AON Hybridisation Assay

To determine the concentration of the AONs (PS229L/PS524 correspondingto SEQ ID NO: 91/92, PS531/PS652 corresponding to SEQ ID NO: 137/185) inplasma and tissue an AON hybridization assay was used, which is based onthe assay described by Yu et al., 2002. For the tissue distributionanalysis, tissues were homogenized, using a MagNaLyzer (Roche) to aconcentration of 60 mg/ml in protK buffer (100 mmol/l Tris-HCl pH8.5,200 mmol/I NaCl, 5 mmol/l EDTA, 0.2% SDS) containing 2 mg/ml proteinaseK, followed by a 2 hours incubation (liver) or 4 hours incubation (allother organs) in a rotating hybridization oven at 55° C. and then stored−20° C. until use. All tissue homogenates and calibration curves werediluted (fit to criteria of the assay) in 60 times diluted pooled mdxcontrol tissue homogenate (kidney, liver, several muscle groups). Atemplate probe specific for each AON (5′ gaatagacg-anti-AON-biotin 3′,DNA phosphate oligonucleotide) and a ligation probe (p-cgtctattc-DIG DNAphosphate oligonucleotide) were used in the hybridization assay. Thehomogenates were incubated for 1 h at 37° C. with template probe (50nmol/1) and the hybridized samples were transferred to streptavidincoated 96-well plates and incubated for 30 min at 37° C. Subsequently,the plate was washed 4 times and the digoxigenin-labeled ligation (2nmol/1) was added and incubated for 30 min at ambient temperature. TheDIG-label was detected using an anti-DIG-POD (1:7,500-1:30,000; RocheDiagnostics), which was visualized with a 3,3′,5,5′-tetramethylbenzidinesubstrate (Sigma Aldrich, the Netherlands), and the reaction was stoppedusing an acidic solution (Sigma Aldrich). The absorption was measured at450 nm using a BioTek Synergy HT plate reader (Beun de Ronde, Abcoude,The Netherlands). Plasma samples were analyzed according to the sameprotocol, using 100 times diluted pooled mdx plasma.

Whole Blood Cytokine Release Assay

For the detection of possible cytokine stimulation induced by selectedAONs (PS232/PS648 corresponding to SEQ ID NO: 119/201 and PS534/PS653corresponding to SEQ ID NO: 139/192) whole blood (anticoagulant CPD)from healthy human volunteers was used. Varying AON concentrations(ranging from 0 to 50 sg/ml, in a dilution of approximately 1:0.01(v/v)) were added to the blood and the samples were incubated for 4hours at 37° C. under 5% CO₂ atmosphere. After incubation, the sampleswere centrifuged at 3200×g for 15 minutes at 4° C. and plasmasupernatants were collected and stored at −20° C. until cytokinequantification. MCP-1, IL-6, TNF-α, and IP-10 concentrations weredetermined by sandwich ELISA (human MCP-1, IL-6, TNF-α, IP-10 ELISA kits(R&D Systems). The experiments with human whole blood were repeatedthree to four times. FIGS. 3A-H are based on one experiment only, butconsidered representative.

Results

The effect on AON activity (i.e., inducing exon skipping efficiency) ofsubstituting all cytosines with 5-methylcytosines (m5C) was tested incultured, differentiated, healthy muscle cells in vitro. In FIGS. 1A and1B two examples are shown. When comparing PS229L and PS524 (=PS229L-m5C)(i.e. non-modified sequence SEQ ID NO: 91 compared with the modifiedsequence SEQ ID NO: 92 wherein all cytosines have been modified) in adose-response transfection experiment using 0-100-200-400 nM, PS524 wasclearly more efficient than PS229L at 200 and 400 nM (1.9-fold higherexon 53 skipping levels) (FIG. 1A). Similarly, when comparing PS220 andPS399 (=PS220-m5C) (i.e. non-modified sequence SEQ ID NO: 101 comparedwith the modified sequence SEQ ID NO: 200 wherein all cytosines havebeen modified) in a dose-response transfection experiment using0-50-100-200-400-800 nM, PS399 was clearly more efficient than PS220,especially at lower concentrations (up to 10-fold higher exon 45skipping levels at 50 nM) (FIG. 1B). These results demonstrate that thepresence of 5-methylcytosines has a positive effect on the activity ofthe AONs. In PS524 (SEQ ID NO:92) all 6 cytosines are substituted with5-methylcytosines (m5C) which had a positive effect on the exon skippingactivity when compared to the non-modified counterpart oligonucleotidePS229L (SEQ ID NO:91) (FIG. 1A). To test whether such positive effectmay be correlated with the number or percentage of base modificationsincorporated, PS1317, PS1318, and PS1319, with respectively 4, 2, and 3of the 6 cytosines substituted with 5-methylcytosines (m5C), were testedand directly compared to PS524 in cultured, differentiated, healthymuscle cells in vitro. PS1317, PS1318, and PS1319 were all effective ininducing exon 53 skipping (47%, 37%, and 45% respectively) (FIG. 1C).When compared to the levels obtained with PS524 however (64%), theseresults indeed suggest that reducing the number of 5-methylcytosines(m5C), from 6 to 4, 3, or 2 5-methylcytosines, leads to a reducedpositive effect on exon skipping activity of the AON.

To investigate whether 5-methylcytosines affect bio-stability,-distribution, and/or -availability, a pharmacokinetic study wasperformed both in wild type (control) and mdx mice. The mdx mouse modelfor DMD has a natural nonsense mutation in exon 23 and is thereforedystrophin-deficient. The lack of dystrophin at the membranes increasesthe permeability of the muscle fibers for relatively small molecules asAONs, and has indeed been demonstrated to enhance 2′-O-methylphosphorothioate RNA AON uptake by muscle up to 10-fold (Heemskerk etal., 2010). The mice were injected subcutaneously with 100 mg/kg ofeither 5-methylcytosine-containing AONs (PS524, PS652 corresponding toSEQ ID NO: 92, 185) or their counterparts with unmodified cytosines(PS229L, PS531 corresponding to SEQ ID NO: 91, 137), three times perweek for two weeks. At different time-points (day 1, 7, 14) after thelast injection, the mice were sacrificed and different muscle groups(heart, diaphragm, gastrocnemius, quadriceps, and triceps) and liver andkidney were isolated to determine AON concentrations therein (FIG. 2A).As anticipated, for all compounds the concentrations in mdx muscles(average of all samples) was higher than those in control mice. Theratio mdx to control AON levels appeared relatively higher for the AONswith 5-methylcytosines. More specifically, in the mdx mice, the levelsof PS524 and PS652 were 2-to 3-fold higher than that of PS229L andPS531. (FIG. 2A). When monitoring the levels of AON in kidney and liver(known toxicity organs), the ratios between muscle tissue and toxicitytissues remained similar, or were even favorable for PS524. Theseresults suggest that AONs with 5-methylcytosine are taken up better byor more stable in muscle than AONs with unmodified cytosines. Indeed thehalf life in muscle was longer for PS524 (>20 days) and PS652 (25 days)when compared to PS229L (7 days) and PS531 (10 days). In plasma, theCmax values of the AONs injected were similar, which confirms that themice received equal doses (FIG. 2B). Remarkably, the AUC values (asindicator for bioavailability) were 1.5 to 2.3-fold higher for the5-methylcytosine containing AONs. This was associated with a lowerclearance which supports their higher muscle tissue levels. The resultsfrom this pharmacokinetic study thus demonstrate that the presence of5-methylcytosines has a positive effect on the bio-stability,-distribution, and/or -availability of the AONs, while themuscle/toxicity organ ratios were similar to those with the AONs withunmodified cytosines.

The in vitro safety profile of AONs with 5-methylcytosines (PS648, PS653corresponding to SEQ ID NO: 201, 192) was compared to that of AONs withunmodified cytosines (PS232, PS534, corresponding to SEQ ID NO: 119,139). AONs may stimulate an innate immune response by activating theToll-like receptors (including TLR7, TLR8, TLR9), which results in setof coordinated immune responses that include innate immunity. Severalchemo- and cytokines, such as IP-10, TNFα, IL-6 and MCP-1 play a role inthis process, and were therefore monitored in human whole bloodincubated with 0 to 50 sg/ml of each AON (using commercially availableELISA kits). PS232 and PS534 both have unmodified cytosines and inducedthe release of TNF-α (FIG. 3A, B), MCP-1 (FIG. 3C, D), IP-10 (FIG. 3E,F), and IL-6 (FIG. 3G, H) at increasing doses. In contrast, both PS648and PS653 (with 5-methylcytosines) did not have any effect on TNF-α,IP-10 and IL-6. PS653, not PS648, seemed to induce a minor release ofMCP-1 only. In conclusion, the presence of 5-methylcytosines improvedthe safety profile of these AONs in vitro.

Example 2 Material and Methods AONs

All oligonucleotides (PS43/PS559/PS1106, all based on SEQ ID NO:31, andcorresponding to SEQ ID NO: 111 (PS43) non modified sequence, SEQ ID NO:202 (PS559) wherein all uraciles have been modified, and SEQ ID NO: 203(PS1106) wherein all uraciles and all cytosines have been modified;PS188/PS785/PS1107, all based on SEQ ID NO:15, and corresponding to SEQID NO: 95 (PS188) non-modified sequence, SEQ ID NO: 204 (PS785) whereinall uraciles have been modified, and SEQ ID NO: 205 (PS1107) wherein alluraciles and all cytosines have been modified; PS235/PS786, both basedon SEQ ID NO:40, and corresponding to SEQ ID NO: 120 (PS235)non-modified sequence and SEQ ID NO: 172 (PS786) wherein all uracileshave been modified), and PS49 (SEQ ID NO:216) non-modified sequence andPS959 (SEQ ID NO:214) wherein all cytosines have been modified, were2′-O-methyl phosphorothioate RNA, and synthesized using an OP-10synthesizer (GE/AKTA Oligopilot) through standard phosphoramiditeprotocols, or obtained from commercial suppliers, in 200 nmol-286 μgscale. Prosensa-synthesized oligonucleotides were cleaved anddeprotected in a two step sequence (DIEA followed by conc. NH₄OHtreatment), purified by HPLC and dissolved in water and an excess ofNaCl was added to exchange ions. After evaporation, compounds wereredissolved in water, desalted by FPLC or ultrafiltration andlyophilized. Mass spectrometry confirmed the identity of all compounds,and purity (determined by UPLC) was found acceptable for all compounds(>75-80%); compounds obtained from commercial sources were used asreceived: PS188 (Girindus, 286.1 g obtained product, purity 93%), PS785,PS786, PS1106, and PS1107 (ChemGenes, 200 nmol synthesis scale, used asreceived), PS43 (Prosensa, 1 μmol synthesis scale, purity 90%), PS559(ChemGenes, 1 μmol synthesis scale, used as received), PS235 (Prosensa,1.92 mmol synthesis scale, purity 91%). For the in vitro transfectionexperiments described herein, 50 μM working solutions of the AONs wereprepared in 20 mM phosphate buffer (pH 7.0).

Transfection and RT-PCR Analysis

Differentiated human healthy control muscle cells (myotubes) weretransfected in 6-wells plates with a fixed AON concentration of 200 nM,according to non-GLP standard operating procedures. For transfectionpolyethylenimine (ExGen500, Fermentas) was used (2 μl per μg AON, in0.15M NaCl). Aforementioned transfection procedures were adapted frompreviously reported material and methods (Aartsma-Rus et al., 2003). At24 hrs after transfection, RNA was isolated and analyzed by RT-PCR.Briefly, to generate dystrophin-specific cDNA, a DMD gene specificreverse primer in exon 53 (PS43/PS559/PS1106, SEQ ID NO: 111, 202, 203),exon 46 (PS188/PS785/PS1107, SEQ ID NO: 95, 204, 205) or exon 54(PS235/PS786, SEQ ID NO: 120, 172) was used in the reverse transcriptase(RT) reaction on 1000 ng input RNA. The PCR analysis was subsequentlydone on 3 μl of dystrophin cDNA for each sample, and included a firstand nested PCR using DMD gene specific primers in exons flanking exon 51(PS43/PS559/PS1106), exon 44 (PS188/PS785/PS1107) or exon 52(PS235/PS786). The RNA isolation and RT-PCR analysis were performedaccording to non-GLP standard operating procedures as described[Aartsma-Rus et al., Hum Mol Genet 2003; 12(8):907-14]. RT-PCR productswere analyzed by gel electrophoresis (2% agarose gels). The resultingRT-PCR fragments were quantified through DNA Lab-on-a-Chip analysis(Agilent). The data was processed by “Agilent 2100 Bioanalyzer” softwareand Excel 2007. The ratio of the smaller transcript product (containingthe exon 51 (PS43/PS559/PS1106), exon 44 (PS188/PS785/PS1107), or exon52 skip (PS235/PS786) to the total amount of transcript products wasassessed (representing the exon 51, 44, or 52 skipping efficiencies inpercentages) and directly compared to that in non-transfected cells.

In Vivo Administration and RT-PCR

The experiments with the mdx mouse model (C57Bl/10ScSn-mdx/J; CharlesRiver Laboratories) were approved by the local LUMC Animal EthicsCommittee (DEC number 11145). Two mdx mice per group were anaesthetizedusing isoflurane and then injected intramuscularly in both gastrocnemiusmuscles, with 20 μg PS49 (SEQ ID NO: 216) or PS959 (SEQ ID NO:214),diluted in sterile saline to a total volume of 50 μl per injection, ontwo consecutive days. Animals were sacrificed 1 week after the lastinjection by cervical dislocation and muscles were isolated and snapfrozen in magnalyzer greenbead tubes (Roche). Six hundred μl Tripure(Roche) was added to the tubes and muscles were homogenized using thebulletblender machine, 3×1 min speed 10. The lysate was transferred to aclean tube to which 120 μl of chloroform was added. Samples werevigorously shaken en incubated on ice for 5 minutes, then centrifugedfor 15 minutes at maximum speed at 4° C. The supernatant was transferredto another tube and 1 volume of isopropanol was added. Samples weremixed and incubated at 4 degrees for at least 30 minutes. Then sampleswere centrifuged for 15 minutes at maximum speed at 4° C., washed with70% ethanol followed by a second centrifugation step of 10 minutes atmaximum speed at 4° C. RNA pellets were air dried and solved in DEPCtreated water. cDNA was generated using 400 ng total RNA with randomhexamer primers using Transcriptor reverse transcriptase (RT) (RocheDiagnostics) according to the manufacturer's instructions. PCRs wereperformed by 30 cycles of 94 degrees for 30 s, 60 degrees for 30 s and72 degrees for 30 s in a 50 μl reaction using 1.5 μl cDNA as templateusing primers specific for mouse exon 22 and exon 24. PCR products werevisualized on 2% agarose gels quantified the Agilent 2100 Bioanalyzer(Agilent, Santa Clara, Calif., USA).

Results

The effect on AON activity (i.e., inducing exon skipping efficiency) ofsubstituting all unmodified cytosines with 5-methylcytosines andsubstituting all unmodified uracils with 5-methyluracils (as in PS1106,PS1107, SEQ ID NO: 203, 205), and of only substituting all unmodifieduracils with 5-methyluracils (as in PS559, PS785, PS786, SEQ ID NO: 202,204, 172), was first tested at a fixed 200 nM AON concentration incultured, differentiated, healthy muscle cells in vitro (FIG. 4A). TheAONs with 5-methyluracils (PS559, PS785, and PS786) increased the exonskipping efficiencies 1.3- to 3-fold when compared to their counterpartswith unmodified uracils. When also replacing the unmodified cytosines by5-methylcytosines, the skipping levels were further increased (PS1106versus PS559, SEQ ID NO: 203 versus 202) or similar (PS1107 versusPS785, SEQ ID NO: 205 versus 204). The effect on AON activity (i.e.inducing exon skipping efficiency) of substituting all unmodifieduracils (as in PS49; SEQ ID NO:216) with 5-methyluracils (as in PS959;SEQ ID NO:214) was then also tested in muscle of the mdx mouse model.PS959 with all 5-methyluracils increased the exon 23 skippingefficiencies approximately 3-fold when compared to PS49 with unmodifieduracils (n=4 per AON) (FIG. 4B). These results demonstrate that not only5-methylcytosines may have a positive effect on exon skipping activity(as also shown in FIGS. 1A-C) but also, 5-methyluracils, both in vitroand in vivo. In addition, the combined use of these 5-methylpyrimidinesmay even further increase activity.

Example 3 Material and Methods AONSs

All oligonucleotides (PS43/PS403, based on SEQ ID NO:31, andcorresponding to SEQ ID NO: 111 (PS43) for the non-modified and SEQ IDNO: 206 (PS403) for the sequence wherein all adenines have beenmodified; PS188/PS733, based on SEQ ID NO:15, and corresponding to SEQID NO: 95 (PS188) for the non-modified and SEQ ID NO: 207 (PS733) forthe sequence wherein all adenines have been modified; PS235/PS897, basedon SEQ ID NO:40, and corresponding to SEQ ID NO: 120 (PS235) for thenon-modified and SEQ ID NO: 173 (PS897) for the sequence wherein alladenines have been modified) were 2′-O-methyl phosphorothioate RNA, andsynthesized using an OP-10 synthesizer (GE/AKTA Oligopilot) throughstandard phosphoramidite protocols, or obtained from commercialsuppliers, in 200 nmol-151 g scale. Prosensa-synthesizedoligonucleotides were cleaved and deprotected in a two step sequence(DIEA followed by conc. NH₄OH treatment), purified by HPLC and dissolvedin water and an excess of NaCl was added to exchange ions. Afterevaporation, compounds were redissolved in water, desalted by FPLC orultrafiltration and lyophilized. Mass spectrometry confirmed theidentity of all compounds, and purity (determined by UPLC) was foundacceptable for all compounds (>75-80%); compounds obtained fromcommercial sources were used as received: PS188 (Girindus, 151 gobtained, purity 92%), PS733 (TriLink or ChemGenes, 200 nmol/1 mgsynthesis scale, used as received, PS43 (Prosensa, 10 μmol synthesisscale, purity 86%), PS403 (ChemGenes, 1 μmol synthesis scale, used asreceived), PS235 (Prosensa, 1.92 mmol synthesis scale, purity 91%),PS897 (ChemGenes, 200 nmol synthesis scale, used as received). For thein vitro transfection experiments described herein, 50 sM workingsolutions of the AONs were prepared in 20 mM phosphate buffer (pH 7.0).For the in vitro complement activation assays described herein, 3 mg/mLstock solutions of PS188 and PS733 were prepared in 20 mM phosphatebuffer (pH 7.0).

Transfection and RT-PCR Analysis

Differentiated human healthy control muscle cells (myotubes) weretransfected in 6-wells plates with a fixed AON concentration of 200 nM,according to non-GLP standard operating procedures. For transfectionpolyethylenimine (ExGen500, Fermentas) was used (2 μl per μg AON, in0.15M NaCl). Aforementioned transfection procedures were adapted frompreviously reported material and methods (Aartsma-Rus et al., 2003). At24 hrs after transfection, RNA was isolated and analyzed by RT-PCR.Briefly, to generate dystrophin-specific cDNA, a DMD gene specificreverse primer in exon 53 (PS43/PS403, SEQ ID NO: 111/206), exon 46(PS188/PS733, SEQ ID NO: 95/207) or exon 54 (PS235/PS897, SEQ ID NO:120/173) was used in the reverse transcriptase (RT) reaction on 1000 nginput RNA. The PCR analysis was subsequently done on 3 l of dystrophincDNA for each sample, and included a first and nested PCR using DMD genespecific primers in exons flanking exon 51 (PS43/PS403), exon 44(PS188/PS733) or exon 52 (PS235/PS897). The RNA isolation and RT-PCRanalysis were performed according to non-GLP standard operatingprocedures as described [Aartsma-Rus et al., Hum Mol Genet 2003;12(8):907-14]. RT-PCR products were analyzed by gel electrophoresis (2%agarose gels). The resulting RT-PCR fragments were quantified throughDNA Lab-on-a-Chip analysis (Agilent). The data was processed by “Agilent2100 Bioanalyzer” software and Excel 2007. The ratio of the smallertranscript product (containing the exon 51 (PS43/PS403), exon 44(PS188/PS733), or exon 52 skip (PS235/PS897) to the total amount oftranscript products was assessed (representing the exon 51, 44, or 52skipping efficiencies in percentages) and directly compared to that innon-transfected cells.

Complement Activation Assay

Antisense oligonucleotides may activate the alternative complementpathway, which contains several split factors, such as C3a and factor Bb(the latter is unique to the alternative pathway). The ability of AONsto possibly activate the complement pathway was assessed in plasma fromCynomolgus monkeys (LiHe plasma, CIT, France). Increasing concentrations(from 0 to 300 μg/mL) of PS188 (SEQ ID NO: 95) and PS733 (PS207), in adilution of 1:10 (v/v)), were added to the plasma and incubated at 37°C. for 30 min. The reaction was terminated by transferring the samplesto ice and making dilutions in ice-cold diluent. Bb and C3aconcentrations were determined by ELISA (Quidel, San Diego, Calif.).

Results

The effect on AON activity (i.e., inducing exon skipping efficiency) ofsubstituting all unmodified adenines with 2,6-diaminopurines was testedat a fixed AON concentration (200 nM) in cultured, differentiated,healthy muscle cells in vitro. In FIG. 5A examples for three differentAON sequences are shown. The AONs with 2,6-diaminopurines (PS403, PS897,and PS733, SEQ ID NO: 206, 207, 173) increased the exon skippingefficiencies 2- to 4-fold when compared to their counterparts withunmodified adenines (compared to SEQ ID NO: 111, 95, 120). There seemedto be a correlation with the number of 2,6-diaminopurines in each AON.

The effect of substituting all unmodified adenines (as in PS188; SEQ IDNO: 95) with 2,6-diaminopurines (as in PS733; SEQ ID NO:207) on in vitrosafety, i.e. possible activation of the alternative complement pathway,was tested in monkey plasma. Whereas PS188 induced relatively highlevels of both split factors Bb and C3a, the 2,6-diaminopurines in PS733completely abolished the effect on the alternative pathway, showing noincrease in either Bb or C3a levels (FIG. 5B). Thus, the presence of2,6-diaminopurines seemed to improve the safety profile of PS188 invitro.

These results demonstrate the positive effect of 2,6-diaminopurines onthe exon skipping activity and safety of AONs.

LIST OF REFERENCES

-   van Ommen, van Deutekom, Aartsma-Rus, Curr Opin Mol Ther. 2008;    10(2):140-9.-   Yokota, Duddy, Partidge, Acta Myol. 2007; 26(3):179-84.-   van Deutekom et al., N Engl J Med. 2007; 357(26):2677-86.-   Goemans et al., N Engl J Med. 2011; 364(16):1513-22.-   Cirak et al., Lancet 2011; 378: 595-605.-   Heemskerk et al., Mol Ther 2010; 18(6):1210-7.-   Aartsma-Rus et al., Hum Mol Gen 2003; 12(8):907-14.-   Yu R Z., Anal Biochem 2002; 304: 19-25.-   Krieg A M. et al., Nature 1995; 374: 546-549.-   Diebold S. S., et. al., Eur J Immunol. 2006; December;    36(12):3256-67.-   Krieg, A. M., Curr. Opin. Immunol. 2000; 12: 35-43.-   Wagner, H., Adv. Immunol. 1999; 73: 329-368.-   Popovic P J. et al. J of Immunol 2006; 177: 8701-8707.-   Peacock H et al. J. Am. Chem. Soc. 2011, 133, 9200-   Arai K et al. Bioorg. Med. Chem. 2011, 21, 6285-   Ehmsen J. et al, J. Cell Sci. 2002, 115 (Pt14): 2801-2803.-   Monaco A. P., et al., Genomics 1988; 2: 90-95.-   Manzur A. Y. et al., Wiley publishers, 2008. The Cochrane    collaboration.-   Hodgetts S., et al, Neuromuscular Disorders 2006; 16: 591-602.-   Aartsma-Rus et al, Oligonucleotides 2010, 20(2): 69-77-   Zuker M., et al, Nucleic Acids Res. 2003; 31(13):3406-15.-   Cartegni L, et al, Nat Rev Genet 2002; 3(4):285-98.-   Cartegni L, et al, Nucleic Acids Res 2003; 31(13):3568-71-   Remington: The Science and Practice of Pharmacy, 20th Edition.-   Baltimore, Md.: Lippincott Williams & Wilkins, 2000-   Kumar L, Pharm. Technol. 2008, 3, 128-   Bruno, K., Advanced Drug Delivery Reviews 2011; 63: 1210.-   Hari et al. Org. Biomol. Chem. 2012, 10, 9639);-   Hanessian et al. Angew. Chem. Intl Ed. 2012, 45, 11242

What is claimed is:
 1. An oligonucleotide comprising a 2′-O-methyl RNAmonomer and a phosphorothioate backbone and comprising a 5-methyluraciland/or a 5-methylcytosine and/or a 2,6-diaminopurine base, wherein saidoligonucleotide is represented by a nucleotide or a base sequencecomprising or consisting of SEQ ID NO:216, or by a nucleotide or a basesequence comprising or consisting of at least a 10 nucleotide contiguousfragment of SEQ ID NO:216, said oligonucleotide having a length of 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32 or 33 nucleotides.
 2. The oligonucleotide of claim 1,wherein the oligonucleotide comprises or consists of a sequence which isreverse complementary to and/or binds to and/or targets and/orhybridizes at least a part of dystrophin pre-mRNA exon
 23. 3. Theoligonucleotide of claim 1, wherein the oligonucleotide has a basesequence comprising or consisting of SEQ ID NO:214 or a base sequencecomprising or consisting of at least a 10 nucleotide contiguous fragmentof SEQ ID NO:214.
 4. A pharmaceutical composition, comprising theoligonucleotide of claim 1 and an excipient.
 5. A method of treating DMDor BMD, comprising administering to a subject having DMD or BMD theoligonucleotide of claim
 1. 6. An oligonucleotide comprising a2′-O-methyl RNA monomer and a phosphorothioate backbone and comprising a5-methyluracil and/or a 5-methylcytosine and/or a 2,6-diaminopurinebase, wherein said oligonucleotide is represented by a nucleotide or abase sequence comprising or consisting of SEQ ID NO:95, or by anucleotide or a base sequence comprising or consisting of at least a 10nucleotide contiguous fragment of SEQ ID NO:95, said oligonucleotidehaving a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 nucleotides.
 7. Theoligonucleotide of claim 6, wherein the oligonucleotide comprises orconsists of a sequence which is reverse complementary to and/or binds toand/or targets and/or hybridizes at least a part of dystrophin pre-mRNAexon
 44. 8. The oligonucleotide of claim 6, wherein the oligonucleotidehas a base sequence comprising or consisting of any one of SEQ IDNOS:204, 205 or 207 or a base sequence comprising or consisting of atleast a 10 nucleotide contiguous fragment of any one of SEQ ID NOS:204,205 or
 207. 9. A pharmaceutical composition, comprising theoligonucleotide of claim 6 and an excipient.
 10. A method of treatingDMD or BMD, comprising administering to a subject having DMD or BMD theoligonucleotide of claim
 6. 11. An oligonucleotide comprising a2′-O-methyl RNA monomer and a phosphorothioate backbone and comprising a5-methyluracil and/or a 5-methylcytosine and/or a 2,6-diaminopurinebase, wherein said oligonucleotide is represented by a nucleotide or abase sequence comprising or consisting of SEQ ID NO:101, or by anucleotide or a base sequence comprising or consisting of at least a 10nucleotide contiguous fragment of SEQ ID NO:101, said oligonucleotidehaving a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 nucleotides.
 12. Theoligonucleotide of claim 11, wherein the oligonucleotide comprises orconsists of a sequence which is reverse complementary to and/or binds toand/or targets and/or hybridizes at least a part of dystrophin pre-mRNAexon
 45. 13. The oligonucleotide of claim 11, wherein theoligonucleotide has a base sequence comprising or consisting of SEQ IDNO:200 or a base sequence comprising or consisting of at least a 10nucleotide contiguous fragment of SEQ ID NO:200.
 14. A pharmaceuticalcomposition, comprising the oligonucleotide of claim 11 and anexcipient.
 15. A method of treating DMD or BMD, comprising administeringto a subject having DMD or BMD the oligonucleotide of claim
 11. 16. Anoligonucleotide comprising a 2′-O-methyl RNA monomer and aphosphorothioate backbone and comprising a 5-methyluracil and/or a5-methylcytosine and/or a 2,6-diaminopurine base, wherein saidoligonucleotide is represented by a nucleotide or a base sequencecomprising or consisting of SEQ ID NO:120, or by a nucleotide or a basesequence comprising or consisting of at least a 10 nucleotide contiguousfragment of SEQ ID NO:120, said oligonucleotide having a length of 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32 or 33 nucleotides.
 17. The oligonucleotide of claim 16,wherein the oligonucleotide comprises or consists of a sequence which isreverse complementary to and/or binds to and/or targets and/orhybridizes at least a part of dystrophin pre-mRNA exon
 52. 18. Theoligonucleotide of claim 16, wherein the oligonucleotide has a basesequence comprising or consisting of SEQ ID NO:172 or 173 or a basesequence comprising or consisting of at least a 10 nucleotide contiguousfragment of SEQ ID NO:172 or
 173. 19. A pharmaceutical composition,comprising the oligonucleotide of claim 16 and an excipient.
 20. Amethod of treating DMD or BMD, comprising administering to a subjecthaving DMD or BMD the oligonucleotide of claim
 16. 21. Anoligonucleotide comprising a 2′-O-methyl RNA monomer and aphosphorothioate backbone and comprising a 5-methyluracil and/or a5-methylcytosine and/or a 2,6-diaminopurine base, wherein saidoligonucleotide is represented by a nucleotide or a base sequencecomprising or consisting of SEQ ID NO:137, or by a nucleotide or a basesequence comprising or consisting of at least a 10 nucleotide contiguousfragment of SEQ ID NO:137, said oligonucleotide having a length of 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32 or 33 nucleotides.
 22. The oligonucleotide of claim 21,wherein the oligonucleotide comprises or consists of a sequence which isreverse complementary to and/or binds to and/or targets and/orhybridizes at least a part of dystrophin pre-mRNA exon
 55. 23. Theoligonucleotide of claim 21, wherein the oligonucleotide has a basesequence comprising or consisting of SEQ ID NO:185 or a base sequencecomprising or consisting of at least a 10 nucleotide contiguous fragmentof SEQ ID NO:185.
 24. A pharmaceutical composition, comprising theoligonucleotide of claim 21 and an excipient.
 25. A method of treatingDMD or BMD, comprising administering to a subject having DMD or BMD theoligonucleotide of claim 21.